Development of DNA probes and immunological reagents specific for cell surface-expressed molecules and transformation-associated genes

ABSTRACT

This invention provides a method for preparing a hybridoma cell line which produces an antibody capable of specifically binding to a cell surface-expressed protein which expresses on the surface of one cell type but not the other. This invention also provides a method for preparing a hybridoma cell line which produces an antibody capable of specifically binding to a cell surface-expressed protein. This invention provides a method to prepare a hybridoma cell line which specifically recognizes and binds to a tumor associated antigen associated with a neoplastic, human cell. This invention also provides a method of preparing DNA encoding a cell surface antigen associated with a neoplastic, human cell. This invention further provides an isolated mammalian nucleic acid molecule having the sequence of Prostate Carcinoma Tumor Antigen Gene-1. This invention also provides an isolated mammalian nucleic acid molecule having the sequence of Prostate Tumor Inducing Gene-1. This invention provides an isolated mammalian nucleic acid molecule having the sequence of Prostate Tumor Inducing Gene-2. Finally, this invention provides an isolated mammalian nucleic acid molecule having the sequence of Prostate Tumor Inducing Gene-1.

The invention disclosed herein was made with Government support underNIH Grants CA 35675 and CA 43208 from the Department of Health and HumanServices. Accordingly, the U.S. Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Throughout this application, various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofeach series of experiments.

The classical method for developing monoclonal antibodies specific forcell-surface molecules involves repeated injections of mice with eitherintact cells or cell membrane preparations derived from the desiredtarget cells. The injections are followed by the removal of mouse spleencells and fusion of these cells to a myeloma partner [reviewed in(1-3)]. This approach has resulted in the production of monoclonalantibodies that react with a number of surface-expressed molecules ofpotential interest, including cell-surface growth factor receptors andtumor-associated antigens. However, the procedure is generallyinefficient and requires screening of a large number of hybridomas forproduction of the appropriate monoclonal antibodies [reviewed in (1-6)].

DNA transfection procedures have been used to transfer human genes intoheterologous cells, such as mouse NIH 3T3 cells (reviewed in 7-10)).When NIH 3T3 cells have been used as the recipient for DNA transfection,this approach has not been successful in identifying dominant-actingtransforming or tumor-inducing genes from a majority (approximately 85%)of human tumors or human tumor cell lines (7-10). In most studies thatuse NIH 3T3 cells, even when a dominant-acting oncogene was identified,it often represented a member of the ras oncogene family or a modifiedcellular gene (7-10). A recently developed cloned rat embryo fibroblastcell line, CREF-Trans 6, has proven useful in identifying putative noveloncogenes not detected in NIH 3T3 cells (11). Cotransfection ofCREF-Trans 6 cells with high-molecular-weight DNA from the LNCaP humanprostatic carcinoma cell line and the selectable neomycin resistancegene (pSV2neo), followed by selection for resistance to G418 andinjection into nude mice, resulted in tumor formation (11). In contrast,when the same DNA sources were used with NIH 3T3 cells, no tumorsdeveloped in nude mice given an injection of neomycin-resistant (G418)cotransfected NIH 3T3 cells (11).

Applicants conducted the current experiments to determine if DNAtransfection combined with an immunologic masking tactic could be usedto efficiently generate hybridomas that secrete monoclonal antibodiesreacting with cell-surface molecules expressed on genetically alteredcells. Applicants demonstrate the feasibility of this approach, calledsurface-epitope masking (SEM). Applicants used DNA transfection and theSEM procedure in an attempt to produce hybridomas secreting monoclonalantibodies that reacted with surface epitopes located on typicalmultidrug-resistant (MDR) cells and human prostatic carcinoma cells.These results indicate that DNA transfection in conjunction with SEM canbe used to generate hybridomas producing monoclonal antibodies that canreact with surface-expressed molecules encoded by both known and unknowngenes.

SUMMARY OF THE INVENTION

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein which expresses on the surface of one celltype but not the other comprises a) generating antiserum against a celltype which does not express the cell surface-expressed protein; b)coating another cell type which expresses the cell surface-expressedprotein with the antiserum generated; c) injecting the antiserum-coatedcells into suitable hosts; d) screening the resulting hosts to identifyhosts which produce serum reactive with the coated cell; e) removingspleens from the hosts-so identified; f) preparing from the spleens soremoved hybridomas; and g) recovering therefrom a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein.

This invention provides a method for isolating DNA coding for a proteincapable of binding to the cell surface-expressed protein which expresseson the surface of one cell type but not the other comprising: a)generating antiserum against a cell type which does not express the cellsurface-expressed protein; b) coating another cell type which expressesthe cell surface-expressed protein with the antiserum generated; c)injecting the antiserum-coated cells into suitable hosts; d) screeningthe resulting hosts to identify hosts which produce serum reactive withthe coated cell; e) removing spleens from the hosts so identified; f)isolating B-lymphocytes from the removed spleen; g) preparing DNA fromplasma cells to generate combinatorial phage cDNA library which containsdifferent clones; and h) contacting the clones in the library with thecoated cells from step (b), the binding of the coated cells with a cloneindicating the protein expressed by the clone capable of binding to thecell surface-expressed protein.

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein which expresses on the surface of one celltype but not the other comprises: a) generating antiserum against a celltype which does not express the cell surface-expressed protein; b)coating another cell type which expresses the cell surface-expressedprotein with the antiserum generated; c) contacting the antiserum-coatedcells with suitable immunoresponsive cells capable of being stimulatedto produce antibodies; d) preparing immunoresponsive cells to producehybridomas; and e) isolating hybridomas which produce antibodiesreactive with the coated cell, thereby preparing hybridoma cell lineswhich produce antibodies capable of specifically binding to a cellsurface-expressed protein.

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein comprises: a) generating antiserum against acell which normally does not express the cell surface-expressed protein;b) introducing a DNA molecule encoding the cell surface-expressedprotein to express the cell surface-expressed protein into the cell; c)selecting cells which express the cell surface-expressed protein; d)coating the selected cells with the antiserum generated in step a; e)injecting the antiserum-coated cells into suitable hosts; f) screeningthe resulting hosts to identify hosts which produce serum reactive withthe coated cell; g) removing spleens from the hosts so identified; h)preparing from the spleens so removed hybridomas; and i) recoveringtherefrom a hybridoma cell line which produces an antibody capable ofspecifically binding to a cell surface-expressed protein.

This invention provides a method for isolating DNA coding for a proteincapable of binding to the cell surface-expressed protein comprising: a)generating antiserum against a cell which normally does not express thecell surface-expressed protein; b) introducing a DNA molecule whichencodes the cell surface-expressed protein to express the cellsurface-expressed protein into the cell; c) selecting cells whichexpress the cell surface-expressed protein; d) coating the selectedcells with the antiserum generated in step a; e) injecting theantiserum-coated cells into suitable hosts; f) screening the resultinghosts to identify hosts which produce serum reactive with the coatedcell; g) removing spleens from the hosts so identified; h) isolatingB-lymphocytes from the removed spleen; i) preparing DNA fromB-lymphocytes to generate combinatorial phage cDNA library whichcontains different clones; and j) contacting the clones in the librarywith the coated cells from step (b), the binding of the coated cellswith a clone indicating the protein expressed by the clone capable ofbinding to the cell surface-expressed protein.

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein which expresses on the surface of one celltype but not the other comprises: a) generating antiserum against a cellwhich normally does not express the cell surface-expressed protein; b)introducing a DNA molecule which encodes the cell surface-expressedprotein to express the cell surface-expressed protein into the cell; c)selecting cells which express the cell surface-expressed protein; d)coating the selected cells with the antiserum generated in step a; e)contacting the antiserum-coated cells with suitable immunoresponsivecells capable of being stimulated to produce antibodies; f) preparingimmunoresponsive cells to produce hybridomas; and g) isolatinghybridomas which produce antibodies reactive with the coated cell,thereby preparing hybridoma cell lines which produce antibodies capableof specifically binding to a cell surface-expressed protein.

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein comprises: a) introducing a DNA molecule whichencodes a cell surface-expressed protein and a second DNA molecule whichencodes a selectable or identifiable trait into an established cellline; b) selecting transfected cells which express the selectable oridentifiable trait; c) recovering the transfected cells so selected; d)coating the selected cells so recovered with an antiserum generatedagainst the established cell line; e) injecting the antiserum-coatedcells into the suitable hosts; f) screening the resulting hosts toidentify hosts which produce serum reactive with the coated cell; g)removing spleens from the hosts so identified; h) preparing from thespleens so removed hybridomas; and i) recovering therefrom a hybridomacell line which produces an antibody capable of specifically binding toa cell surface-expressed protein.

This invention provides a method for isolating DNA coding for a proteincapable of binding to the cell surface-expressed protein comprising: a)introducing a DNA molecule which encodes a cell surface-expressedprotein and a second DNA molecule which encodes a selectable oridentifiable trait into an established cell line; b) selectingtransfected cells which express the selectable or identifiable trait; c)recovering the transfected cells so selected; d) coating the selectedcells so recovered with an antiserum generated against the establishedcell line; e) injecting the antiserum-coated cells into the suitablehosts; f) screening the resulting hosts to identify hosts which produceserum reactive with the coated cell; g) removing spleens from the hostsso identified; h) isolating B-lymphocytes from the removed spleen; i)preparing DNA from B-lymphocytes to generate combinatorial phage cDNAlibrary which contains different clones; and j) contacting the clones inthe library with the coated cells from step (b), the binding of thecoated cells with a clone indicating the protein expressed by the clonecapable of binding to the cell surface-expressed protein.

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein comprises a) introducing a DNA molecule whichencodes a cell surface-expressed protein and a second DNA molecule whichencodes a selectable or identifiable trait into an established cellline; b) selecting transfected cells which express the selectable oridentifiable trait; c) recovering the transfected cells so selected; d)coating the selected cells so recovered with an antiserum generatedagainst the established cell line; e) contacting the antiserum-coatedcells with suitable immunoresponsive cells capable of being stimulatedto produce antibodies; f) preparing immunoresponsive cells to producehybridomas; and g) isolating hybridomas which produce antibodiesreactive with the coated cell of step (d), thereby preparing hybridomacell lines which produce antibodies capable of specifically binding to acell surface-expressed protein.

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody which specifically recognizes and binds to atumor associated antigen associated with a neoplastic, human cell whichcomprises: a) cotransfecting the CREF-Trans 6 cell line (ATCC AccessionNo. CRL 10584) with DNA isolated from a neoplastic, human cell and DNAwhich encodes a selectable or identifiable trait; b) selectingtransfected cells which express the selectable or identifiable trait; c)recovering the transfected cells so selected; d)injecting thetransfected cells so recovered into a suitable first murine host; e)maintaining the resulting first murine host for a period of timeeffective to induce the injected transfected cells to form a tumor inthe first murine host; f) isolating the resulting tumor from the firstmurine host; g) obtaining tumor cells from the tumor so isolated; h)coating the tumor cells so obtained with an antiserum generated againstthe established non-human, non-tumorigenic cell line; i) injecting theantiserum-coated cells into the suitable second hosts; j) screening theresulting second hosts to identify hosts which produce serum reactivewith the neoplastic, human cell; k) removing spleens from the secondhosts so identified;l) preparing from the spleens so removed hybridomas;and m) recovering therefrom a hybridoma cell line which produces anantibody which specifically recognizes and binds to the cell surfaceantigen.

This invention provides a method of preparing DNA encoding a cellsurface antigen associated with a neoplastic, human cell whichcomprises: a) cotransfecting CREF-Trans 6 cell line with DNA isolatedfrom a neoplastic human cell and DNA encoding a selectable oridentifiable trait; b) selecting transfected cells which express theselectable or identifiable trait; c) recovering the transfected cells soselected; d) injecting the transfected cells so recovered into asuitable first murine host; e) maintaining the resulting first murinehost for a period of time effective to induce the injected transfectedcells to form a tumor in the first murine host; f) isolating theresulting tumor from the first murine host; g) obtaining tumor cellsfrom the tumor so isolated; and h) recovering DNA encoding the cellsurface antigen associated with the neoplastic human cell from the tumorcells so obtained.

This invention further provides an isolated mammalian nucleic acidmolecule having the sequence of Prostate Carcinoma Tumor Antigen Gene-1.This invention also provides an isolated mammalian nucleic acidmolecules having the sequence of Prostate Tumor Inducing Gene-1. Thisinvention provides an isolated mammalian nucleic acid molecules havingthe sequence of Prostate Tumor Inducing Gene-2. Finally, this inventionprovides an isolated mammalian nucleic acid molecules having thesequence of Prostate Tumor Inducing Gene-3.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Flow chart for SEM strategy. This procedure involves theproduction of anti-CREF-Trans 6 polyclonal antibodies, which are used toblock rat antigenic epitopes on transfected CREF-Trans 6 prior toinjection into animals. The strategy results in the production of immunespleen cells that react with transfected surface-expressed antigens onCREF-Trans 6 cells. Spleen cells are then fused with NS1 murine myelomacells, producing hybridomas secreting monoclonal antibodies specific forantigens expressed on the cell surface of transfected CREF-Trans 6cells.

FIG. 2 Reactivity toward MDR CREF-Trans 6 cells subject to SEM producedMDR monoclonal antibodies. CREF-Trans 6 cells were transfected with ahuman MDR-1 gene and MDR clones resistant to colchicine were isolated.The SEM approach was used to generate independent hybridomas (MDR 2.3,MDR 3.0, MDR 8.12 and MDR 9.7) that secreted monoclonal antibodiesreacting with surface epitopes of the MDR transporter expressed inCREF-Trans 6:MDR A1 cells but not untransfected non-MDR CREF-Trans 6parental cells. The SEM-derived monoclonal antibodies were also testedfor reactivity with three independently derived CREF-Trans 6 MDR clones.CREF-Trans 6:MDR C3, CREF-Trans 6:MDR D2 and CREF-Trans 6:MDR F4.Samples were analyzed by FACS, and results are expressed as meanfluorescence intensity units. Replicate samples varied by <10% andreplicate studies varied by <20%.

FIG. 3 Reactivity toward MCF7 and MDR MCF7 cells of human leukocyteantigen class 1 monoclonal antibodies and MDR monoclonal antibodiesproduced using CREF-Trans 6:MDR A1 cells and the SEM procedure. MCF7 andMCF7 CL4 cells are non-MDR cells. MCF7 CL4:MDR I, MCF7 CL4:MDR II, andMCF7 CL4:MDR III are three independent MDR MCF7 CL4 subclones.Fluorescence baseline was determined using irrelevant isotype-matchedantibody and goat anti-mouse immunoglobulin G conjugated to fluorescineisothiocyanate. Samples were analyzed by FACS and results are expressedas mean fluorescence intensity units. Replicate samples varied by ≦10%and replicate studies varied by ≦20%.

FIG. 4 Reactivity toward untransfected CREF-Trans 6, LNCaPDNA-transfected CREF-Trans 6 and human prostatic carcinoma cell lines ofSEM-derived monoclonal antibodies. Pro 1.1, 1.2, 1.3, 1.4 and 1.5 aremonoclonal antibodies produced by independent hybridomas generated byfusing spleen cells from mice immunized with LNCaP DNA-transfected,tumor-derived CREF-Trans 6 cells coated with anti-CREF-Trans 6polyclonal antibodies, CREF-Trans 6:4 NMT, with NS1 murine myelomacells. The cells analyzed were parental CREF-Trans 6-4 NMT (primaryLNCaP DNA-transfected, tumor derived transfectant derived from tumor),CREF-Trans 6:4-7 NMT (secondary CREF-Trans 6:4 NMT DNA-transfected,tumor-derived transfectant derived from tumor), and human prostaticcarcinoma cell lines LNCaP, DU-145 and PC-3. Samples were analyzed byFACS, and results are expressed as mean fluorescence intensity units.Replicate samples varied by <10% and replicate studies varied by <15%.

FIG. 5 Expression of putative human prostatic carcinoma encodedpolypeptides in LNCaP DNA-transfected tumor-derived CREF-Trans 6 cellsand LNCaP and DU-145 human prostatic carcinoma cells. Labeled celllysates from the cell lines were immunoprecipitated with monoclonalantibody Pro 1.4. Molecular weight size markers are indicated on theleft side of the figure. Experimental details can be found in theMaterials and Methods section and Duigou et al. (20).

FIG. 6 Differential display (DD) of CREF-Trans 6 and CREF-Trans 6:4 NMTmRNAs. DD was performed with a 24 oligomer (5′-) and a 14 oligomer (3′-)with sequences shown in materials and methods. The arrow indicates thePTI-1 band appearing only in mRNA from CREF-Trans 6:4 NMT, but not inCREF-Trans 6 cells. The length of this PTI-1 DNA fragment is 214 bp.

FIG. 7 Expression of PTI-1 in normal and tumor cell lines. RNAs fromCREF-Trans 6, CREF-Trans 6:4 NMT and various normal and tumor-derivedhuman cell lines were transferred to nylon membranes and probed with a[32P]-labeled 279 bp PTI-1 DNA fragment (317 to 596 bp, between primersA and L, FIG. 8). Membranes were stripped and reprobed with a[³²P]-labeled GAPDH gene.

FIG. 8 Peptide and DNA sequence of the PTI-1 gene and comparison withthe human EF-1α gene. (A) Peptide and DNA sequence of PTI-1. The 5′- and3′-non-translation region of the PTI-1 gene is in small letters and thePTI-1 open-reading frame is in capital letters. Squared amino acids aremutated amino acids in the PTI-1 gene resulting from single-basemutations (underlined bases). The sequences underlined in the5′-non-translated regions are the L and A primers, used in FIGS. 7 and9. (B) Peptide comparison of EF-1α and PTI-1. Peptide for EF-1α isindicated in (E) and the peptide for PTI-1 is indicated in (P) Theunderlined region (67 amino acids) of EF-1α indicates the amino acidsmissing in the PTI-1 gene. Bold letters (with a *) indicate the mutatedamino acids in the PTI-1 peptide. (C) Differences in amino acids, codonsand nucleotides between EF-1α and PTI-1. Six single-base mutations giverise to specific amino acid changes in the PTI-1 gene. In the column ofamino acids, the numbers in parentheses refer to the position of theamino acid in the peptide and codon refers to the sequence of threenucleotides encoding the specific amino acid. The specific nucleotidechange is also indicated.

FIG. 9 RT-PCR analysis of PTI-1, PSA and GAPDH expression in cell linesand tissue samples of normal prostate, BPH and prostate carcinoma.RT-PCR of PTI-1 uses two primers consisting of a pair of 20-oligomers:primer L with the sequences 5′-GAGTCTGAATAGGGCGACTT-3′ (senseorientation); and primer A with the sequence 5′-AGTCAGTACAGCTAGATGCC-3′(antisense orientation) (both underlined in FIG. 8). RT-PCR of PSA usesthe primers (A) 5′-AGACACAGGCCAGGTATTTCAGGTC-3′ and (B)5′-CACGATGGTGTCCTTGATCCACTTC-3′. RT-PCR of GAPDH uses a pair of primerswith the sequences (I) (5′-TCTTACTCCTTGGAGGCCATG-3′) and P (II)(5′-CGTCTTCACCACCATGGAGAA-3′). The PCR amplified products were blottedon nylon membranes and probed with a [³²P]-labeled 279 bp DNA fragmentof PTI-1, PSA or GAPDH, respectively.

FIG. 10 In vitro translation of the PTI-1 gene. Lane CAT is the in vitrotranslation of the chloramphenicol acetyltransferase gene (M_(r)=24kDa), used as a positive control. Lane PTI-1 contains the translatedproducts of the PTI-1 gene. Rainbow protein standards (Amersham LifeScience) were used to determine the sizes of the in vitro translatedproducts.

FIG. 11 Expression of PTI-1 in CREF cells transformed by differentoncogenes. Northern hybridization analysis of RNA isolated from:CREF-Trans 6; LNCaP DNA transfected nude mouse tumor-derived CREF-Trans6 cells (CREF-Trans 6:4 NMT); LNCaP; CREF cells transformed by a mutantof type 5 adenovirus (CREF-H5hr1/A2); CREF cells transformed by adexamethasone inducible (mouse mammary tumor virus (MMTV) promoter) wildtype 5 adenovirus transforming E1A gene (CREF/MMTV-Ad5E1A) in theabsence of DEX (-DEX) normal cellular phenotype, in the presence of DEX(+DEX) Ad5 E1A expressed and cells are transformed; CREF cellstransformed by Ha-ras oncogene (CREF-ras); CREF cells transformed byv-src oncogene (CREF-src); and CREF cells transformed by oncogenic humanpapilloma virus type 51 (CREF-HPV-51). Blots probed with a ³²P-labeledPTI-1 gene probe, then stripped and reprobed with a ³²P-labeled GAPDHgene probe.

FIG. 12 Reactivity of Br-car (breast carcinoma) monoclonal antibodies(MAbs) prepared by the surface epitope masking (SEM) technique towardfresh-frozen sections of human cancers. Sections were prepared frompatients with metastatic melanoma (A), small cell lung carcinoma (B) andbreast carcinomas (C and D). Reactivity was determined usingimmunohistochemical techniques with MAbs prepared using the SEMprocedure with nude mouse tumor-derived CREF-Trans 6 cells transfectedwith DNA from the human breast carcinoma cell line T47D, CREF-Trans6:T47D NMT. Reactivity is only apparent in the two human breastcarcinoma sectioned patient samples.

FIG. 13 Nucleic acid sequence of PTI-2

FIG. 14 Nucleic acid sequence of PTI-3

FIG. 15 Nucleic acid sequence of PCTA-1

FIG. 16 Immunofluorescence staining and immunoprecipitation analysis ofsecreted and cellular PCTA-1. Live cells were incubated with MoAb Pro1.5 and analyzed by fluorescence microscopy (A, B, C). Cells werelabeled with ³⁵S-methionine and secreted and cellular PCTA-1 levels weredetermined by immunoprecipitation with the Pro 1.5 MoAb (D).

FIG. 17 Immunohistochemical detection of PCTA-1 and prostate specificantigen (PSA) in prostate tissue sections. Benign prostate (PCTA-1 (A),PSA (E)], prostatic intraepithelial neoplasia (PIN) [PCTA-1 (C), PSA(F)] and invasive prostate carcinoma [PCTA-1 (C and D), PSA (G and H)].Original magnifications: A, B, D, E, F and H=250X; C and G=100X.

FIG. 18 Predicted amino acid sequence of PCTA-1 and alignment of PCTA-1with other galactose-binding lectin (galectin) proteins. The predictedamino acid sequence is shown below the nucleotide sequence (A).Comparison of PCTA-1 with sequences of the M_(r) 14,000 and M_(r) 29,000to 31,000 galectins from eel, chicken, mouse, rat, bovine and humangalectins (B). *, amino acids unique to PCTA-1; _, amino acids shared byPCTA-1 and mouse-L34, human galectin-3-L29 and human-L31; o, amino acidsshared by PCTA-1 and the M_(r) 14,000 and M_(r) 29,000 to 31,000galectins from different species.

FIG. 19 RT-PCR analysis of PCTA-1, PSA and GAPDH expression in celllines and tissue samples of normal prostate, BPH, and prostatecarcinoma. RT-PCR of PCTA-1 uses two 20-mers with the sequences5′-AAGCTGACGCCTCATTTGCA-3′ and 5′-AACCACCAATGGAACTGGGT-3′. RT-PCR of PSAuses two primers: primer A, 5′-AGACACAGGCCAGGTATTTCAGGTC-3′; and primerB, 5′-CACGATGGTGTCCTTGAT CCACTTC-3′. RT-PCR of GAPDH uses a pair ofprimers with the sequences 5′-TCTTACTCCTTGGAGGCCATG-3′ and5′-CGTCTTCACCACCATGGAGAA-3′. The PCR-amplified products were blotted onnylon membranes and probed with a ³²P-labeled DNA fragment of PCTA-1,PSA, or GAPDH, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, the following standard abbreviations areused throughout the specification to indicate specific nucleotides:

-   -   C=cytosine A=adenosine    -   T=thymidine G=guanosine

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein which expresses on the surface of one celltype but not the other comprises a) generating antiserum against a celltype which does not express the cell surface-expressed protein; b)coating another cell type which expresses the cell surface-expressedprotein with the antiserum generated; c) injecting the antiserum-coatedcells into suitable hosts; d) screening the resulting hosts to identifyhosts which produce serum reactive with the coated cell; e) removingspleens from the hosts so identified; f) preparing from the spleens soremoved hybridomas; and g) recovering therefrom a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein.

This invention provides a method for isolating DNA coding for a proteincapable of binding to the cell surface-expressed protein which expresseson the surface of one cell type but not the other comprising: a)generating antiserum against a cell type which does not express the cellsurface-expressed protein; b) coating another cell type which expressesthe cell surface-expressed protein with the antiserum generated; c)injecting the antiserum-coated cells into suitable hosts; d) screeningthe resulting hosts to identify hosts which produce serum reactive withthe coated cell; e) removing spleens from the hosts so identified; f)isolating B-lymphocytes from the removed spleen; g) preparing DNA fromplasma cells to generate combinatorial phage cDNA library which containsdifferent clones; and h) contacting the clones in the library with thecoated cells from step (b), the binding of the coated cells with a cloneindicating the protein expressed by the clone capable of binding to thecell surface-expressed protein.

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein which expresses on the surface of one celltype but not is the other comprises: a) generating antiserum against acell type which does not express the cell surface-expressed protein; b)coating another cell type which expresses the cell surface-expressedprotein with the antiserum generated; c) contacting the antiserum-coatedcells with suitable immunoresponsive cells capable of being stimulatedto produce antibodies; d) preparing immunoresponsive cells to producehybridomas; and e) isolating hybridomas which produce antibodiesreactive with the coated cell, thereby preparing hybridoma cell lineswhich produce antibodies capable of specifically binding to a cellsurface-expressed protein.

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein comprises: a) generating antiserum against acell which normally does not express the cell surface-expressed protein;b) introducing a DNA molecule encoding the cell surface-expressedprotein to express the cell surface-expressed protein into the cell; c)selecting cells which express the cell surface-expressed protein; d)coating the selected cells with the antiserum generated in step a; e)injecting the antiserum-coated cells into suitable hosts; f) screeningthe resulting hosts to identify hosts which produce serum reactive withthe coated cell; g) removing spleens from the hosts so identified; h)preparing from the spleens so removed hybridomas; and i) recoveringtherefrom a hybridoma cell line which produces an antibody capable ofspecifically binding to a cell surface-expressed protein.

This invention provides a method for isolating DNA coding for a proteincapable of binding to the cell surface-expressed protein comprising: a)generating antiserum against a cell which normally does not express thecell surface-expressed protein; b) introducing a DNA molecule whichencodes the cell surface-expressed protein to express the cellsurface-expressed protein into the cell; c) selecting cells whichexpress the cell surface-expressed protein; d) coating the selectedcells with the antiserum generated in step a; e) injecting theantiserum-coated cells into suitable hosts; f) screening the resultinghosts to identify hosts which produce serum reactive with the coatedcell; g) removing spleens from the hosts so identified; h) isolatingB-lymphocytes from the removed spleen; i) preparing DNA fromB-lymphocytes to generate combinatorial phage cDNA library whichcontains different clones; and j) contacting the clones in the librarywith the coated cells from step (b), the binding of the coated cellswith a clone indicating the protein expressed by the clone capable ofbinding to the cell surface-expressed protein.

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein which expresses on the surface of one celltype but not the other comprises: a) generating antiserum against a cellwhich normally does not express the cell surface-expressed protein; b)introducing a DNA molecule which encodes the cell surface-expressedprotein to express the cell surface-expressed protein into the cell; c)selecting cells which express the cell surface-expressed protein; d)coating the selected cells with the antiserum generated in step a; e)contacting the antiserum-coated cells with suitable immunoresponsivecells capable of being stimulated to produce antibodies; f) preparingimmunoresponsive cells to produce hybridomas; and g) isolatinghybridomas which produce antibodies reactive with the coated cell,thereby preparing hybridoma cell lines which produce antibodies capableof specifically binding to a cell surface-expressed protein.

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein comprises: a) introducing a DNA molecule whichencodes a cell surface-expressed protein and a second DNA molecule whichencodes a selectable or identifiable trait into an established cellline; b) selecting transfected cells which express the selectable oridentifiable trait; c) recovering the transfected cells so selected; d)coating the selected cells so recovered with an antiserum generatedagainst the established cell line; e) injecting the antiserum-coatedcells into the suitable hosts; f) screening the resulting hosts toidentify hosts which produce serum reactive with the coated cell; g)removing spleens from the hosts so identified; h) preparing from thespleens so removed hybridomas; and i) recovering therefrom a hybridomacell line which produces an antibody capable of specifically binding toa cell surface-expressed protein.

This invention provides a method for isolating DNA coding for a proteincapable of binding to the cell surface-expressed protein comprising: a)introducing a DNA molecule which encodes a cell surface-expressedprotein and a second DNA molecule which encodes a selectable oridentifiable trait into an established cell line; b) selectingtransfected cells which express the selectable or identifiable trait; c)recovering the transfected cells so selected; d) coating the selectedcells so recovered with an antiserum generated against the establishedcell line; e) injecting the antiserum-coated cells into the suitablehosts; f) screening the resulting hosts to identify hosts which produceserum reactive with the coated cell; g) removing spleens from the hostsso identified; h) isolating B-lymphocytes from the removed spleen; i)preparing DNA from B-lymphocytes to generate combinatorial phage cDNAlibrary which contains different clones; and j) contacting the clones inthe library with the coated cells from step (b), the binding of thecoated cells with a clone indicating the protein expressed by the clonecapable of binding to the cell surface-expressed protein.

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody capable of specifically binding to a cellsurface-expressed protein comprises a) introducing a DNA molecule whichencodes a cell surface-expressed protein and a second DNA molecule whichencodes a selectable or identifiable trait into an established cellline; b) selecting transfected cells which express the selectable oridentifiable trait; c) recovering the transfected cells so selected; d)coating the selected cells so recovered with an antiserum generatedagainst the established cell line; e) contacting the antiserum-coatedcells with suitable immunoresponsive cells capable of being stimulatedto produce antibodies; f) preparing immunoresponsive cells to producehybridomas; and g) isolating hybridomas which produce antibodiesreactive with the coated cell of step (d), thereby preparing hybridomacell lines which produce antibodies capable of specifically binding to acell surface-expressed protein.

In an embodiment, the DNA molecules are introduced into the establisedcell line by cotransfection.

In another embodiment, the DNA molecule encoding a cellsurface-expressed protein is a expression vector.

In another embodiment, the cell surface-expressed protein is theP-glycoprotein.

In another embodiment, the cell surface-expressed protein is a cytokinereceptor.

In a further embodiment, the cytokine receptor is a interferon-alphareceptor.

In still another embodiment, the cytokine receptor is a interferon-gammareceptor.

In yet another embodiment, the cell surface-expressed protein is a tumorassociated antigen.

In a still further embodiment, the second DNA molecule encoding theselectable or identifiable trait is plasmid DNA.

In another embodiment, the plasmid DNA encodes resistance to anantibiotic.

In still another embodiment, the plasmid DNA comprises pSV2-Neo.

In another embodiment, the cell line is the CREF-Trans 6 cell line (ATCCAccession No. CRL 10584).

This invention provides a method for preparing a hybridoma cell linewhich produces an antibody which specifically recognizes and binds to atumor associated antigen associated with a neoplastic, human cell whichcomprises: a) cotransfecting the CREF-Trans 6 cell line (ATCC AccessionNo. CRL 10584) with DNA isolated from a neoplastic, human cell and DNAwhich encodes a selectable or identifiable trait; b) selectingtransfected cells which express the selectable or identifiable trait; c)recovering the transfected cells so selected; d) injecting thetransfected cells so recovered into a suitable first murine host; e)maintaining the resulting first murine host for a period of timeeffective to induce the injected transfected cells to form a tumor inthe first murine host; f) isolating the resulting tumor from the firstmurine host; g) obtaining tumor cells from the tumor so isolated; h)coating the tumor cells so obtained with an antiserum generated againstthe established non-human, non-tumorigenic cell line; i) injecting theantiserum-coated cells into the suitable second hosts; j) screening theresulting second hosts to identify hosts which produce serum reactivewith the neoplastic, human cell; k) removing spleens from the secondhosts so identified;l) preparing from the spleens so removed hybridomas;and m) recovering therefrom a hybridoma cell line which produces anantibody which specifically recognizes and binds to the cell surfaceantigen.

In an embodiment, the neoplastic, human cell is a benign cell. Inanother embodiment, the neoplastic, human cell is a metatastic cell. Ina separate embodiment, the neoplastic, human cell is a human prostaticcarcinoma cell derived from cell line LNCaP (ATCC No. CRL 1740). Inanother embodiment, the cell is a human breast carcinoma cell derivedfrom cell line T47D (ATCC No. HTB 133).

In an embodiment, the suitable second host is a murine host. In anotherembodiment, the suitable second host is a non-human primate host.

This invention provides a method of preparing DNA encoding a cellsurface antigen associated with a neoplastic, human cell whichcomprises: a) cotransfecting CREF-Trans 6 cell line with DNA isolatedfrom a neoplastic human cell and DNA encoding a selectable oridentifiable trait; b) selecting transfected cells which express theselectable or identifiable trait; c) recovering the transfected cells soselected; d) injecting the transfected cells so recovered into asuitable first murine host; e) maintaining the resulting first murinehost for a period of time effective to induce the injected transfectedcells to form a tumor in the first murine host; f) isolating theresulting tumor from the first murine host; g) obtaining tumor cellsfrom the tumor so isolated; and h) recovering the DNA encoding the cellsurface antigen associated with the neoplastic human cell from the tumorcells so obtained. The DNA molecule containing the sequence for the cellsurface antigen associated with the neoplastic human cell may be furtherisolated.

In an embodiment, the neoplastic, human cell is a benign tumor cell. Inanother embodiment, the neoplastic, human cell is a metastatic cell. Ina separate embodiment, the neoplastic, human cell is a human prostaticcarcinoma cell derived from cell line LNCaP (ATCC No. CRL 1740). Inanother embodiment, the neoplastic, human cell is a human breastcarcinoma cell derived from cell line T47D (ATCC No. HTB133). In anotherembodiment, the neoplastic, human cell is a human glioblastoma multiform(stage IV astrocytoma) cell derived from a primary tumor. In a furtherembodiment, the neoplastic, human cell is a patient-derived metastaticcolon carcinoma.

In a separate embodiment, the DNA encoding the selectable oridentifiable trait is plasmid DNA encoding resistance to an antibiotic.In a further embodiment, the plasmid DNA comprises pSV2-Neo and theselection is by the antibiotic G418.

The cell surface antigen of the above-described method includes but notlimited to a tumor associated antigen, a growth factor receptor, aviral-encoded surface-expressed antigen, a oncogene product, a surfaceepitope, a membrane protein which mediates classical multi-drugresistance, a membrane protein which mediates atypical multi-drugresistance, an antigen which mediates a tumorigenic phenotype, anantigen which mediates a metastatic phenotype, an antigen whichsuppresses a tumorigenic phenotype, an antigen which suppresses ametastatic phenotype and cytokine receptors including the humaninterferon α and interferon γ receptors.

In an embodiment, the cell surface antigen is an antigen which isrecognized by a specific immunological effector cell. In a furtherembodiment, the specific immunological effector cell is a T-cell.

In a separate embodiment, the cell surface antigen is an antigen whichis recognized by a non-specific immunological effector cell. In afurther embodiment, the non-specific immunological effector cell is amacrophage cell. In a still further embodiment, the non-specificimmunological effector cell is a natural killer cell.

This invention provides the DNA prepared the above-described method.This invention also provides nucleic acid probes hybridizable with theisolated DNA molecule. The nucleic acid probe may be DNA or RNA. In anembodiment, the nucleic acid probe is labeled with a detectable marker.In a further embodiment, the DNA probe is labeled with a detectablemarker.

This invention also provides a method of diagnosing in a subject aneoplastic condition which comprises contacting a sample from thesubject with the above-described DNA probe under conditions permittingthe DNA probe to hybridize with the DNA associated with the neoplasticcondition, detecting the presence of hybridized DNA, and therebydiagnosing the neoplastic condition.

This invention also provides monoclonal antibody designated Pro 1.1.;monoclonal antibody designated Pro 1.2.; monoclonal antibody designatedPro 1.3.; monoclonal antibody designated Pro 1.4.; and monoclonalantibody designated Pro 1.5.

This invention provides an isolated mammalian nucleic acid moleculehaving the sequence of Prostate Carcinoma Tumor Antigen Gene-1. Thenucleic acid molecule may be DNA, cDNA or RNA. This invention alsoprovides isolated human nucleic acid molecule.

The nucleic acid molecules described and claimed herein are useful forthe information which they provide concerning the amino acid sequence ofthe polypeptide and as products for the large scale synthesis of thepolypeptide by a variety of recombinant techniques. The molecule isuseful for generating new cloning and expression vectors, transformedand transfected prokaryotic and eukaryotic host cells, and new anduseful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

This invention also provides nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence ofProstate Carcinoma Tumor Antigen Gene-1.

This nucleic acid molecule produced can either be DNA or RNA. As usedherein, the phrase “specifically hybridizing” means the ability of anucleic acid molecule to recognize a nucleic acid sequence complementaryto its own and to form double-helical segments through hydrogen bondingbetween complementary base pairs.

This nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a sequence of a Prostate Carcinoma TumorAntigen Gene-1 can be used as a probe. Nucleic acid probe technology iswell known to those skilled in the art who will readily appreciate thatsuch probes may vary greatly in length and may be labeled with adetectable label, such as a radioisotope or fluorescent dye, tofacilitate detection of the probe. DNA probe molecules may be producedby insertion of a Prostate Carcinoma Tumor Antigen Gene-1 DNA moleculeinto suitable vectors, such as plasmids or bacteriophages, followed bytransforming into suitable bacterial host cells, replication in thetransformed bacterial host cells and harvesting of the DNA probes, usingmethods well known in the art. Alternatively, probes may be generatedchemically from DNA synthesizers.

RNA probes may be generated by inserting the Prostate Carcinoma TumorAntigen Gene-1 DNA molecule downstream of a bacteriophage promoter suchas T3, T7 or SP6. Large amounts of RNA probe may be produced byincubating the labeled nucleotides with the linearized DNA fragmentwhere it contains an upstream promoter in the presence of theappropriate RNA polymerase.

This invention provides an isolated mammalian nucleic acid moleculehaving the sequence of Prostate Carcinoma Tumor Antigen Gene-1operatively linked to a promoter of RNA transcription.

This invention also provides vectors which comprises the isolatedmammalian nucleic acid molecule having the sequence of ProstateCarcinoma Tumor Antigen Gene-1. In an embodiment, the vector is aplasmid.

This invention also provides the plasmid designated PCTA-1. Thisplasmid, PCTA-1, was deposited on Jan. 11, 1995 with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852,U.S.A. under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganism for the Purposes of PatentProcedure. Plasmid, PCTA-1, was accorded ATCC Accession Number 97201.

PCTA-1 gene contains 3850 bp. It was cloned into the XhoI and EcoRIsites of the pBluescript vector. T3 promoter is close to 5′ end and T7promoter to 3′ end of PCTA-1.

This invention also provides an antisense oligonucleotide having asequence capable of specifically hybridizing to an mRNA moleculeencoding the Prostate Carcinoma Tumor Antigen Gene-1 protein so as toprevent translation of the mRNA molecule.

This invention also provides methods of detecting expression of theProstate Carcinoma Tumor Antigen Gene-1 in a sample which contains cellscomprising steps of: (a) obtaining total RNA from the cells; (b)contacting the RNA so obtained with a labelled nucleic acid molecule ofclaim 7 under hybridizing conditions; and (c) determining the presenceof RNA hybridized to the molecule, thereby detecting the expression ofthe Prostate Carcinoma Tumor Antigen Gene-1 in the sample.

This invention provides methods of detecting expression of the ProstateCarcinoma Tumor Antigen Gene-1 in tissue sections which comprises stepsof: (a) contacting the tissue sections with a labelled nucleic acidprobe under hybridizing conditions permitting hybridization of the probeand the RNA of Prostate Carcinoma Tumor Antigen Gene-1; and (b)determining the presence of RNA hybridized to the probe, therebydetecting the expression of the Prostate Carcinoma Tumor Antigen Gene-1in tissue sections.

This invention also provides the above-described isolated mammaliannucleic acid molecule operatively linked to a promoter of RNAtranscription.

This invention also provides a vector which comprises theabove-described isolated mammalian nucleic acid molecule. In anembodiment, the vector is a plasmid. In another embodiment, the vectoris a virus. In a further embodiment, the virus is a DNA virus. In astill further embodiment, the virus is an RNA virus. In anotherembodiment, the virus is a retrovirus.

This invention provides purified mammalian Prostate Carcinoma TumorAntigen Gene-1 protein. This invention also provides polypeptidesencoded by the above-described isolated mammalian nucleic acidmolecules.

This invention also provides antibodies capable of specifically bindingto the mammalian Prostate Tumor Inducing Gene-1 protein. This inventionalso provides an antibody capable of competitively inhibiting thebinding of the above antibodies with the mammalian Prostate TumorInducing Gene-1 protein. In an embodiment, these antibodies aremonoclonal.

This invention also provides a pharmaceutical composition comprising theabove-described antibody and a pharmaceutically acceptable carrier. Thisinvention also provides a therapeutic agent comprising theabove-described antibodies and a cytotoxic agent. The cytotoxic agentmay be a radioisotope or toxin.

This invention provides methods for measuring the amount of a mammalianProstate Carcinoma Tumor Antigen Gene-1 protein in a biological samplecomprising steps of: a) contacting the biological sample with thespecific antibody directed to the mammalian Prostate Carcinoma TumorAntigen Gene-1 protein under the condition permitting the formation of acomplex with said antibody and the mammalian Prostate Carcinoma TumorAntigen Gene-1 protein, and b) measuring the amount of said complex,thereby measuring the amount of the Prostate Carcinoma Tumor AntigenGene-1 protein in said biological sample. In an embodiment, the sampleis a serum sample.

This invention also provides methods to determining whether a subjectcarries a cancer with metastatic potential comprising steps of: (a)measuring the amount of Prostate Carcinoma Tumor Antigen Gene-1 proteinin the serum sample of the subject; and (b) comparing the amountdetermined in step (a) with the amount determined from the sample of ahealthy subject.

This invention provides methods for determining whether a compound iscapable of inhibiting the expression of Prostate Carcinoma Tumor AntigenGene-1 protein comprising steps of: (a) contacting the transformed cellsof claim 19 with an appropriate amount of the compound under conditionspermitting the compound to inhibit expression of Prostate CarcinomaTumor Antigen Gene-1 protein; and (b) detecting the level of theProstate Carcinoma Tumor Antigen Gene-1 protein expression, a decreasein the expression level indicating that the compound is capable ofinhibiting the expression of Prostate Carcinoma Tumor Antigen Gene-1protein.

This invention provides methods for determining whether a compound iscapable of specifically binding to the Prostate Carcinoma Tumor AntigenGene-1 protein comprising steps of:(a) contacting an appropriate amountof the purified Prostate Carcinoma Tumor Antigen Gene-1 protein with anappropriate amount of the compound under conditions permitting formationof a complex between the compound and the purified protein; (b)detecting such complex, the presence of the complex indicating that thecompound is capable of binding to the receptor.

This invention provides pharmaceutical compositions comprising aneffective amount of the compound capable of either binding or inhibitingthe activity of the Prostate Carcinoma Tumor Antigen Gene-1 protein asdetermined by the above-methods and a pharmaceutical carrier.

This invention also provides a method of treating cancer with metastaticpotential in a subject comprising administering effective amount of theabove pharmaceutical composition, therapeutic agent alone or incombination of, to the subject.

This invention provides an isolated mammalian nucleic acid moleculehaving the sequence of Prostate Tumor Inducing Gene-1. The nucleic acidmolecule can be DNA, cDNA, genomic DNA, synthetic DNA or RNA. Thisinvention also provides human nucleic acid molecule.

This invention further provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence ofProstate Tumor Inducing Gene-1.

This invention provides an isolated mammalian nucleic acid moleculehaving the sequence of Prostate Tumor Inducing Gene-1 operatively linkedto a promoter of RNA transcription.

This invention provides a method of detecting expression of a ProstateTumor Inducing Gene-1 in a cell which comprises obtaining total mRNAfrom the cell, contacting the mRNA so obtained with a labelled nucleicacid molecule of at least 15 nucleotides capable of specificallyhybridizing with a sequence of Prostate Tumor Inducing Gene-1 underconditions permitting hybridization, and determining the presence ofmRNA hybridized to the molecule, thereby detecting the expression of theProstate Tumor Inducing Gene-1 in the cell.

In one embodiment of this invention, nucleic acids are extracted byprecipitation from lysed cells and the mRNA is isolated from the extractusing an oligo-dT column which binds the poly-A tails of the mRNAmolecules. The mRNA is then exposed to a radioactively labelled probe ona nitrocellulose membrane, and the probe hybridizes to and therebylabels complementary mRNA sequences. Binding may be detected byluminescence autoradiography or scintillation counting. However, othermethods for performing these steps are well known to those skilled inthe art, and the discussion above is merely an example.

This invention also provides a method of detecting expression of aProstate Tumor Inducing Gene-1 in tissue sections which comprisescontacting the tissue sections with a labelled nucleic acid molecule ofat least 15 nucleotides capable of specifically hybridizing with asequence of Prostate Tumor Inducing Gene-1 under hybridizing conditions,determining the presence of mRNA hybridized to the molecule, and therebydetecting the expression of the Prostate Tumor Inducing Gene-1 in tissuesections.

The probes are also useful for in-situ hybridization or in order tolocate tissues which express this gene, or for other hybridizationassays for the presence of this gene or its mRNA in various biologicaltissues. The in-situ hybridization using a labelled nucleic acidmolecule is well known in the art. Essentially, tissue sections areincubated with the labelled nucleic acid molecule to allow thehybridization to occur. The molecule will carry a marker for thedetection because it is “labelled”, the amount of the hybrid will bedetermined based on the detection of the amount of the marker and sowill the expression of the Prostate Tumor Inducing Gene-1.

This invention provides an isolated mammalian nucleic acid moleculehaving the sequence of the Prostate Tumor Inducing Gene-1 operativelylinked to a promoter of RNA transcription.

The isolated mammalian nucleic acid molecule having the sequence of theProstate Tumor Inducing Gene-1 can be linked to vector systems. Variousvectors including plasmid vectors, cosmid vectors, bacteriophage vectorsand other viruses are well known to ordinary skilled practitioners.

This invention also provides vectors which comprises the isolatedmammalian nucleic acid molecule having the sequence of the ProstateTumor Inducing Gene-1. In an embodiment, the vector is a plasmid.

In an embodiment, the Prostate Tumor Inducing Gene-1 sequence is clonedin EcoRI/XhoI site of the Bluescript vector. This plasmid, PTI-1, clone18, was deposited on Jan. 11, 1995 with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A.under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganism for the Purposes of PatentProcedure. Plasmid, PTI-1, was accorded ATCC Accession Number 97020.

PTI-I gene is determined by RACE from 1 to 215 bp and the rest by PTI-1,clone. The plasmid PTI-1 is clone 18, contains 1937 bp (29 bp+1908 bp)insert in pBluescript vector EcoRI (5′ end of insert) and XhoI (3′ polyAside).

The 1937 bp insert can be cut out by restriction enzymes XhoI+EcoRI. 5′end of insert is close to T3 promoter, 3′ end is close to T7 promoter.

Experiments demonstrate that the 29 bp sequence of insert comes from thesecondary structure of RNA so that this 29 bp sequence was not shown incomplete sequence of PTI-I gene (see FIG. 8), and replaced by the rightsequence obtained from RACE method.

The first 29 bp sequence is:5′CGGCCCGAGCTCGTGCCGAATTCGGCCCGAGAGCGTTAAAGTGTGATGGCGTA CATCTT. Thesequence from 30-1937 bp (1907 bp) is the sequence 216-2,123 bp (1907bp) in complete sequence of PTI-1 (FIG. 8).

As an example to obtain these vectors, insert and vector DNA can both beexposed to a restriction enzyme to create complementary ends on bothmolecules which hybridize with each other and are then ligated togetherwith DNA ligase. Alternatively, linkers can be ligated to the insert DNAwhich correspond to a restriction site in the vector DNA, which is thendigested with the restriction enzyme which cuts at that site. Othermeans to obtain the vectors are also available and known to an ordinaryskilled practitioner.

This invention provides a host vector system for the production of apolypeptide having the biological activity of a mammalian Prostate TumorInducing Gene-1 protein which comprises the above described vectors anda suitable host. These vectors may be transformed into a suitable hostcell to form a host cell vector system for the production of apolypeptide.

Regulatory elements required for expression include promoter sequencesto bind RNA polymerase and transcription initiation sequences forribosome binding. For example, a bacterial expression vector includes apromoter such as the lac promoter and for transcription initiation theShine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryoticexpression vector includes a heterologous or homologous promoter for RNApolymerase II, a downstream polyadenylation signal, the start codon AUG,and a termination codon for detachment of the ribosome. Such vectors maybe obtained commercially or assembled from the sequences described bymethods well known in the art, for example, the methods described abovefor constructing vectors in general.

This invention also provides a method of producing a polypeptide havingthe biological activity of a mammalian Prostate Tumor Inducing Gene-1protein which comprises growing the host cells of the above describedhost vector system under suitable conditions permitting production ofthe polypeptide and recovering the polypeptide so produced.

This invention also provides mammalian cells comprising theabove-described nucleic acid molecule.

This invention provides purified mammalian Prostate Tumor InducingGene-1 protein. This invention also provides a polypeptide encoded bythe isolated mammalian nucleic acid molecule having the sequence ofProstate Tumor Inducing Gene-1 protein.

This invention also provides a method to produce an antibody using theabove-described mammalian Prostate Tumor Inducing Gene-1 protein.

This invention provides an antibody capable of binding specifically tothe mammalian Prostate Tumor Inducing Gene-1 protein. In an embodiment,the antibody is a monoclonal antibody.

This invention also provides a therapeutic agent comprising theabove-described antibody and a cytoxic agent. In an embodiment, thecytotoxic agent is either a radioisotope or toxin.

This invention further provides an immunoassay for measuring the amountof a mammalian Prostate Tumor Inducing Gene-1 protein in a biologicalsample comprising steps of: a) contacting the biological sample with atleast one of the above-described antibodies to form a complex with saidantibody and the mammalian Prostate Tumor Inducing Gene-1 protein; andb) measuring the amount of the Prostate Tumor Inducing Gene-1 protein insaid biological sample by measuring the amount of said complex.

This invention also provides a method of inactivating oncogenictransformation of cells comprising inactivating the expression of theexpression of the 5′-UTR of Prostate Tumor Inducing Gene-1. In anembodiment, the the inactivation is carried out by deleting the completeor a portion of the 5′-UTR sequence.

This invention provides an isolated mammalian nucleic acid moleculehaving the sequence of Prostate Tumor Inducing Gene-2. The nucleic acidmolecule can be DNA, cDNA, genomic DNA, synthetic DNA or RNA. Thisinvention also provides human nucleic acid molecule.

This invention further provides a nucleic acid molecule of at least 15nucleotides capable of specifically is hybridizing with a sequence ofProstate Tumor Inducing Gene-2.

This invention provides an isolated mammalian nucleic acid moleculehaving the sequence of Prostate Tumor Inducing Gene-2 operatively linkedto a promoter of RNA transcription.

This invention also provides vectors which comprises the isolatedmammalian nucleic acid molecule having the sequence of Prostate TumorInducing Gene-2. In an embodiment, the vector is a plasmid.

This invention also provides the plasmid designated PTI-2. This plasmid,PTI-2, was deposited on Jan. 11, 1995 with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A.under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganism for the Purposes of PatentProcedure. The plasmid, PTI-2, was accorded with ATCC Accession Number69742.

PTI-2 contains 1819 bp DNA. It was cloned into the XhoI and EcoRI siteof the pBluescript vector. T3 promoter is close to the 5′-end and T7promoter to the 3′-end of PTI-2. THe (1819 bp) insert of PTI-2 can becut out with XhoI and EcoRI enzyme.

This invention provides an isolated mammalian nucleic acid moleculehaving the sequence of Prostate Tumor Inducing Gene-3. The nucleic acidmolecule can be DNA, cDNA, genomic DNA, synthetic DNA or RNA. Thisinvention also provides human nucleic acid molecule.

This invention further provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence ofProstate Tumor Inducing Gene-3.

This invention provides an isolated mammalian nucleic acid moleculehaving the sequence of Prostate Tumor Inducing Gene-3 operatively linkedto a promoter of RNA transcription.

This invention also provides vectors which comprises the isolatedmammalian nucleic acid molecule having the sequence of Prostate TumorInducing Gene-3. In an embodiment, the vector is a plasmid.

This invention also provides the plasmid designated PTI-3. This plasmid,PTI-3, was deposited on Jan. 11, 1995 with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A.under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganism for the Purposes of PatentProcedure. Plasmid, PTI-3, was accorded ATCC Accession Number 97022.

PTI-3 is a partial sequence of the gene. A 1869 bp DNA of PTI-3 isinserted into PCR™II vector (3.9 kb). 5′-end of the insert is adjacentto Sp-6 promoter and 3′-end is adjacent to T7 promoter.

The insert can be cut out with EcoRI restriction enzyme to obtain the1869 bp DNA (with extra 5 bp vector sequence at both sides).

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

First Series of Experiments

Materials and Methods

Cell Types and Culture Conditions

The CREF-Trans 6 cells line (11) is a specific subclone of the FischerF2408 rat embryo fibroblast (CREF) cell line that displays an increasedsensitivity for expression of transfected genes compared with parentalCREF cells (12). The LNCaP cell line was derived from metastases from apatient with advanced prostate cancer (13) and was provided by Dr. A. K.Ng (Department of Pathology, Columbia University, College of Physiciansand Surgeons, New York, N.Y.). and Dr. Steven Harris (W. Alton JonesCell Science Center, New York, N.Y.). CREF-Trans 6:4 NMT cells werederived from a tumor induced in a nude mouse following injection ofG418-resistant CREF-Trans 6 cells cotransfected with DNA from LNCaPcells and a dominant-acting neomycin resistance gene (pSV2neo) (11).CREF-Trans 6:4-7 NMT cells were derived from a tumor induced in a nudemouse following injection of G418-resistant CREF-Trans 6 cellscontransfected with DNA from CREF-Trans 6:4 NMT cells and the pSV2neoplasmid (11). MDR CREF-Trans 6 cells were produced by transfectingCREF-Trans 6 cells with an expression vector plasmid containing a humanMDR (also known as PGY1) gene (pHaMDR1/A) (14), which was provided byDr. Michael M. Gottesman (National Cancer Institute, Bethesda, Md.) andselecting for colchicine resistance as previously described (15).

For this study, four independent CREF-Trans 6 MDR clones were used: 1)CREF-Trans 6:MDR Al, 2) CREF-Trans 6:MDR C3, 3) CREF-Trans 6:MDR D2, and4) CREF-Trans 6:MDR F4. All four CREF-Trans 6 MDR clones displayedincreased resistance to colchicine versus parental CREF-Trans 6 cells,and they were cross-resistant to vincristine, doxorubicin, anddactinomycin (data not shown). The human prostatic carcinoma cell linesDU-145 and PC-3 were obtained from the American Type Culture Collection(Rockville, Md.) The human breast carcinoma cell line MCF7 was providedby Dr. John W. Greiner (National Cancer Institute). MCF7 CL4 C1 (MCF7CL4) is a single-cell-derived subclone of MCF7 established in one ofapplicants' laboratories at Columbia University. MDR MCF7 CL4 subclones(MCF7 CL4:MDR 1, MCF7 CL4:MDR II and MCF7 CL4:MDR III) were obtained ina manner similar to that used for CREF-Trans 6 MDR clones. MCF7 CL4:MDRI, MCF7 CL4:MDR II and MCF7 CL4:MDR III cells contained MDR1 messengerRNA (mRNA), expressed the 170-kd P-glycoprotein, and displayed increasedresistance to colchicine and vincristine compared with MCF7 and MCF7 CL4cells (data not shown). GBM-18 tumor cells were derived from a patientwith a stage IV astrocytoma (glioblastoma multiforme) (16). Normal humanskin fibroblasts, NHSF-1, were established from a skin biopsy andprovided by Dr. Armand F. Miranda (Department of Pathology, ColumbiaUniversity) (17). The human melanoma cell line H0-1 was provided from a49-year-old woman and was provided by Dr. Beppino Giovanella (StehlinFoundation for Cancer Research, Houston, Tex.) (18). The human melanomacell line-MeWo was provided by Dr. Robert S. Kerbel (Sunnybrook HealthScience Center, Toronto, Canada). The human colon carcinoma cell linesWiDr and LS174T were provided by Dr. John W. Greiner.

CREF-Trans 6, CREF-Trans 6 MDR subclone, CREF-Trans 6:4 NMT, CREF-Trans6:4-7 NMT, H0-1 and MeWo cells were grown, at 37° C. in Dulbecco'smodified Eagle's medium supplemented with 5% fetal bovine serum. Normalhuman skin fibroblasts (NHSF-1) as well as the LNCaP, WiDr, LS174T, MCF7and MCF7 CL4 cells and the MCF7 CL4 MDR subclone cells were grown inDulbecco's modified Eagle's medium supplemented with 10% fetal bovineserum. All cells were maintained in the logarithmic phase of growth byculturing at a 1:5 or 1:10 ratio of resuspended cells to fresh mediumprior to confluence.

Preparation of Mouse Polyclonal Antibodies, Enzyme-Linked ImmunosorbentAssay, and SEM

BALB/c female mice (8-10 weeks old) (Charles River BreedingLaboratories, Wilmington, Mass.) were hyperimmunized with CREF-Trans 6cells. Their care was in accordance with institutional guidelines.Animals received one subcutaneous injection of manually resuspendedCREF-Trans 6 cells mixed with complete Freund's adjuvant (1:1) on day 0.On day 7, animals received a subcutaneous injection of manuallyresuspended CREF-Trans 6 cells mixed with incomplete Freund's adjuvant(1:1). On days 14 and 21, animals received an intraperitoneal injectionof manually resuspended CREF-trans 6 cells in Hanks' phosphate-bufferedsolution. On day 35, mice were bled from the retro-orbital eye socket,and the sera were prepared and tested for anti-CREF-Trans 6 activity byenzyme-linked immunosorbent assay. For these assays, CREF-Trans 6 cellswere grown in 96-well microtiter plates to near confluence. Cells werefixed with 3.7% formalin in phosphate-buffered solution (5 minutes atroom temperature) and blocked with 10% normal goat serum (60 minutes at37° C.). The anti-CREF-Trans 6 antisera were titered against fixedCREF-Trans 6 cells (serial dilutions of antisera, 2 hours at 37° C.).Binding to CREF-Trans 6 cells was detected using a goat anti-mouseimmunoglobulin secondary antibody conjugated to horseradish peroxidase(60 minutes at 37° C.). 3,3′, 5,5′. tetramethylbenzidine (Kirkegaard andPerry, Gaithersburg, Md.) was added in the presence of H₂O₂ and positivebinding was monitored by a color change and quantitated byspectrophotometer (19).

In this study, the SEM procedure involved the coating of transfectedCREF-Trans 6 cells with high-titer mouse anti-CREF-Trans 6 antisera toblock the rat antigenic molecules prior to hyperimmunizing BALB/c mice.One to 3 million transfected CREF-Trans 6 cells were incubated overnightat 4° C. with a 1:100 dilution of mouse anti-CREF-Trans 6 antisera.Prior to the injection of polyclonal antibody-coated transfectedCREF-Trans 6 cells into BALB/c mice, cells were first incubated in 1%neutral-buffered formalin for 5 minutes at 4° C. Mice were given fourinjections of formalin-fixed cells over a 21-day period using a protocolsimilar to that utilized for developing mouse anti-CREF-Trans 6antisera. The spleens of hyperimmunized mice were removed, and spleencells were isolated and fused with NS1 murine myeloma cells (AmericanType Culture Collection) to form hybridomas as previously described(19).

Immunoprecipitation Analysis

Immunoprecipitation analysis was performed as described previously (20).CREF-Trans 6, CREF-Trans 6:4 NMT, CREF-Trans 6:4-7 NMT, LNCaP, andDU-145 cells were grown to 80% confluence in 6-cm plates, starved ofmethionine for 1 hour at 37° C. in methionine-free medium (20) andlabeled for 2 hours at 37° C. in 1 mL of the same medium with 1 mCi of[³⁵S] methionine (Express ³⁵S ; NEN Chemicals, Boston, Mass.). Celllysates were prepared and immunoprecipitated with the Pro-1.4 monoclonalantibody (produced by hybridomas prepared using the SEM procedure withCREF-Trans 6:4 NMT cells) as described previously (20).

Fluorescence-Activated Cell Sorter Analysis

Fluorescence-activated cell sorter (FACS) analysis was performed asdescribed previously (21, 22). Results are expressed as meanfluorescence intensity units. Monoclonal antibodies specific for humanleukocyte antigen class I antigens were supplied by Dr. Soldano Ferrone(New York Medical College, Valhalla). All studies were performed aminimum of three times with duplicate samples in each experiment.Replicate samples within individual experiments varied 10% or less, andthe variation between experiments was generally 20% or less.

Experimental Results

Development of Monoclonal Antibodies Reacting With MDR CREF-Trans 6 andMCF7 Cells

To determine the feasibility of the SEM approach for developingmonoclonal antibodies reactive with cell-surface antigens on transfectedtarget cells, applicants performed initial studies using a definedmolecule expressed on the cell surface, i.e., the typical MDR geneproduct. A schematic of the SEM protocol is shown in FIG. 1.Overexpression of the MDR1 gene results in an increased quantity of the170-kd membrane glycoprotein (P-glycoprotein), which functions as anadenosine triphosphate-dependent drug efflux pump [reviewed in (23)].CREF-Trans 6 cells were transfected with the pHaMDR1/A expression vector(14), and clones surviving in colchicine were isolated. CREF-Trans 6:MDRclones produced MDR1 mRNA, expressed the 170-kd P-glycoprotein, anddisplayed cross-resistance to other chemotherapeutic agents, includingvincristine, doxorubicin, and dactinomycin (data not shown). An MDRCREF-Trans 6 clone (i.e., CREF-Trans 6:MDR A1) was used in combinationwith the SEM procedure to generate monoclonal antibodies specific forthe MDR P-glycoprotein. CREF-Trans 6:MDR A1 cells were coated withpolyclonal antibody produced against CREF-Trans 6 cells fixed informalin and injected four times over a 21-day period into BALB/c mice.Spleen cells were isolated and fused with the NS1 murine myeloma cellline, resulting in hybridomas secreting monoclonal antibodies reactingwith outer epitopes of the P-glycoprotein on additional independentlyderived CREF-Trans 6:MDR clones (FIG. 2). The four monoclonal antibodiesspecific for the P-glycoprotein, MDR 2.3, MDR 3.6, MDR 8.12 and MDR 9.7,reacted with CREF-Trans 6:MDR A1, CREF-Trans 6:MDR C3, CREF-Trans 6:MDRD2, and CREF-Trans 6:MDR F4 cells. In contrast, the differentSEM-derived MDR monoclonal antibodies did not react with a large numberof non-MDR cells, including CREF-Trans 6, CREF-Trans 6:4 NMT, LNCaP,MCF7, WiDr, LS174T, H0-1, MeWo, NHSF-1 or GBM-18 (data not shown).

Applicants then determined if an additional cell type expressing thesame MDR1 gene and the MDR phenotype as CREF-Trans 6:MDR A1 cells alsocontained the same P-glycoprotein surface antigenic epitopes. MDR MCF7CL4 cells were developed by transfection with pHaMDR1/A and selectionfor colchicine resistance. MCF7 parental cells and thesingle-cell-derived MCF7 subclone, MDF7 CL4, did not display the MDRphenotype and did not react with monoclonal antibodies MDR 2.3, MDR 3.6,MDR 8.12, or MDR 9.7 (FIG. 3). However, both MCF7 and MCF7 CL4 cellsreacted with human leukocyte antigen class I monoclonal antibodies. Aseries of independently derived MDR MCF7 CL4 subclones, including MCF7CL4:MDR I, MCF7 CL4:MDR II and MCF7 CL4:MDR III cells, was found toreact with both the human leukocyte antigen class I and SEM-derived MDRmonoclonal antibodies (FIG. 3). These results indicate that monoclonalantibodies developed using the SEM approach with transfected CREF-Trans6 cells can also react with additional cell types expressing the samesurface-localized molecules.

Development of Monoclonal Antibodies Reacting With Human ProstaticCarcinoma Cells

Cotransfection of CREF-Trans 6 cells with high-molecular-weight DNA fromthe human prostatic carcinoma cell line LNCaP and pSV2neo plasmid,followed by selection for G418 resistance and injection into nude mice,results in tumor formation (11). To determine if tumor-derivedCREF-Trans 6 cells display novel surface molecules related to theoriginal transforming human tumor DNA, applicants used the SEM procedurewith a primary nude mouse tumor-derived cell line, CREF-Trans 6:4 NMT(11). Cells were coated with CREF-Trans 6 polyclonal antibodies, fixedin formalin, and injected repeatedly into BALB/c mice. As describedabove for MDR CREF-Trans 6 cells, hybridomas were produced, and specifichybridomas were identified that produced monoclonal antibodies reactingwith both primary tumor-derived (CREF-Trans 6:4 NMT) and secondarytumor-derived (CREF-Trans 6:4-7 NMT) cells (FIG. 4). These monoclonalantibodies, designated Pro 1.1, Pro 1.2, Pro 1.3, Pro 1.4 and Pro 1.5did not react by FACS analysis with CREF-Trans 6, NHSF-1, GBM-18, WiDr,LS174T, MeWo, or H0-1 cells (data not shown). All five monoclonalantibodies did, however, react with LNCaP cells, and specific Promonoclonal antibodies also reacted (as demonstrated by FACS analysis)with two additional human prostatic carcinoma cell lines, PC-3 andDU-145.

The degree of surface binding of the different Prc monoclonal antibodiesto the same cell type varied, suggesting that these monoclonalantibodies may recognize different epitopes on the same tumor-associatedantigen. With the majority of the Pro monoclonal antibodies, binding wasgreater with LNCaP cells than with CREF-Trans 6:4 NMT or CREF-Trans6:4-7 NMT cells. In the case of PC-3 and DU-145 human prostaticcarcinoma cells, four (1.2, 1.3, 1.4 and 1.5) of the five Pro monoclonalantibodies bound to PC-3 cells, whereas low-level binding was apparentonly with Pro 1.2, 1.4 and 1.5 in DU-145 cells. Preliminary FACSanalysis also indicated that Pro 1.1, 1.3 and 1.5 displayed significantbinding to the surface of two human breast carcinoma cell lines, T47Dand MCF7 (data not shown).

To obtain additional information about the Pro monoclonal antibodiesgenerated using the SEM approach, applicants performedimmunoprecipitation analysis of polypeptides encoded by transfectedCREF-Trans 6 and human prostatic carcinoma cells (FIG. 5). Cells werelabeled with [³⁵S] methionine, and cell lysates were prepared andcombined with monoclonal antibody Pro-1.4. Immunoprecipitates wereanalyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(19). A protein of approximately 42 kd was immunoprecipitated from celllysates produced from CREF-Trans 6:4 NMT, CREF-Trans 6:4-7 NMT, LNCaP,and DU-145 (FIG. 5). In contrast, this potentially new tumor-associatedantigen was not detected in cell lysates obtained from CREF-Trans 6,NHSF-1, GBM-18, WiDR, MeWo, or H0-1 cells (FIG. 5 and data not shown).The relative quantity of the immunoprecipitated 42 kd tumor-associatedantigen was greatest in CREF Trans 6:4-7 NMT and LNCaP cells which alsodisplayed the highest level of surface binding using monoclonal antibodyPro 1.4 and FACS analysis (FIG. 4). These results indicate that the SEMprocedure can be used to generate monoclonal antibodies recognizingsurface-expressed human antigens expressed on transfected CREF-Trans 6cells containing unidentified human transforming genes that encodetumor-associated antigens expressed on the cell surface.

Experimental Discussion

In many classes of neoplastic cells, unique sets of tumor-associatedantigens are present that are not expressed or are expressed at lowerlevels compared with normal cellular counterparts [reviewed in (2-6)].The classical approach for detecting these molecules is to use intactcells or cell membrane preparations from tumor cell lines or primarytumor samples to generate hybridomas, producing monoclonal antibodiesreacting with specific tumor-associated antigens [reviewed in (1-3, 6)].This approach is laborious and often unsuccessful in generatingmonoclonal antibodies that display the necessary specificity to permittheir use for cancer diagnostics or therapeutics (3, 4, 6). Analternative strategy, which is dependent upon a functional changeinduced in a target cell, involves the use of DNA transfection and anapproach termed “surface-epitope masking.” In the present study,applicants demonstrate the utility of this combined strategy for thegeneration of monoclonal antibodies specific for a known surfaceexpressed molecule, the P-glycoprotein-mediating MDR, and the product ofan unidentified putative human prostatic carcinoma gene. The SEMapproach has also been used to produce monoclonal antibodies reactingwith the human γ-interferon receptor (Su, Z-z., Pestka, S., Fisher, P.B.: manuscript in preparation) and an unidentified putative human breastcarcinoma gene (Yemul, S., Su, Z-z., Leon, J. A., et al: manuscript inpreparation) expressed in CREF-Trans 6 cells. All of these resultsindicate that the combination of transfection and SEM offers a uniqueopportunity to generate monoclonal antibodies specific for humantumor-associated antigens without prior knowledge of the identity of thegene encoding these products. With appropriate expression vector geneconstructs, this strategy can also be used to generate monoclonalantibodies reacting with well-characterized, surface-localized proteinsexpressed in CREF-Trans 6 cells. This combined approach, at least inprinciple, should be applicable to any experimental model in whichspecific changes occur in the expression of surface molecules between a“tester” (a transfected cell expressing a new surface-expressedmolecule) and a “driver” (the untransfected parental cell).

The identity and function of the putative tumor-inducing human prostaticcarcinoma gene that has been stably transferred from LNCaP cells toCREF-Trans 6 cells are not known. However, CREF-Trans 6:4 NMT cells thatcontain this potential prostatic carcinoma gene can be used to generatemonoclonal antibodies reactive with surface antigens on bothLNCaP-transfected cells and human prostatic carcinoma cell lines. Thisability suggest a potential causal relationship between expression ofthe transfected gene and expression of the prostatic carcinomaphenotype.

Prior studies (24, 25) have indicated that transformed NIH 3T3 cellstransfected with specific human tumor DNAs could be used to generatemonoclonal antibodies specific for surface antigens expressed by theoriginal tumor cell line as well as histologically similar tissue types.This approach has been used to generate monoclonal antibodies specificfor cell-surface antigens expressed on NIH 3T3 cells transformed byhuman pancreatic carcinoma (24) and acute myelogenous leukemia (25) DNA.The monoclonal antibodies produced against DNA-transformed NIH 3T3 cellsfrom human pancreatic carcinoma reacted with surface antigens ontransfected transformed cells, the original human pancreatic carcinomacell line, and six additional human pancreatic carcinoma cell lines(24). These monoclonal antibodies did not react with untransfected NIH3T3 cells or human lymphoblastoid, melanoma, prostatic carcinoma, ornormal human skin fibroblast cell lines (24). Results from both studies(24, 25) indicate that the combination of DNA transfection andmonoclonal antibody development may prove useful in generatingmonoclonal antibodies with specificity for cell-surface epitopesdisplayed by different histologic tumor types. In this respect, theability of CREF-Trans 6 to identify tumor-inducing genes withoutbiological activity in NIH 3T3 cells indicates that this new human tumorDNA transfection-acceptor cell line may prove useful for theidentification and cloning of potentially novel genetic elementsmediating specific human cancers.

In the present study, the SEM approach was used to stimulate theproduction of spleen cells reactive with cell-surface-accessiblemolecules. Spleen cells were then used to produce hybridomas thatsecrete monoclonal antibodies reactive with accessible surface antigens.The SEM approach described in this manuscript uses a formalin fixationstep prior to immunizing animals with polyclonal antibody-coated testercells. This procedure was originally adopted to more efficiently producemonoclonal antibodies that would have direct diagnostic potential, i.e.,they could be used to detect antigens on formalin-fixed tissue.Monoclonal antibodies produced using the SEM procedure have demonstratedspecificity for viable cells, frozen tissue specimens, and bothformalin-fixed tissue specimens and formalin-fixed cells. The SEMprocedure described in this application would not be predicted togenerate monoclonal antibodies reactive with fixative-sensitiveantigens. However, modifications of the SEM approach using proceduresother than formalin fixation, including injection of unfixedantibody-coated cells or use of novel immune complexes (26), shouldresult in the generation of monoclonal antibodies reacting with fixativesensitive antigenic epitopes of molecules expressed on the cell surface.

Recently, an approach called “phage display combinatorial libraries” hasbeen developed in which combinatorial complementary DNA (cDNA) librariesare prepared in phage directly from antigen-stimulated spleen cells (27,28). In this context, transfected cells that had been subject to the SEMprocedure could be utilized as immunogens to stimulate an antigenicresponse that could then be followed by the isolation of spleen cellsand generation of combinatorial phage cDNA libraries. This approachcould then result in the direct identification of potentially criticalgenes involved with transformation and with genes that encode specifichuman tumor-associated antigens and other molecules expressed on thecell surface.

The SEM approach has been performed using murine monoclonal antibodiesto coat rat antigenic surface epitopes on a rat embryo fibroblast cellline, CREF-Trans 6. Alternatively, it should be possible to usetransfected CREF-Trans 6 cells as direct immunogens in syngeneic Fischerrats for the generation of rat hybridomas or rat x mouse heteromyelomas.Although these studies are still in progress, it is apparent that theSEM approach employing murine monoclonal antibody-coated CREF-Trans 6 ispreferable to injection of transfected CREF-Trans 6 cells directly intosyngeneic rats. Murine monoclonal antibodies are relatively easy toproduce and are highly amenable to purification in large quantities. Inaddition, the technologies required for the genetic manipulation ofmurine monoclonal antibodies (e.g., chimerization, humanization, andbispecific monoclonal antibodies) are readily available (29-31). Thesegenetic approaches are extremely important if a monoclonal antibody isto be used in human clinical trials for imaging or as a therapeuticagent (4-6).

The theoretical basis of the SEM approach involves antigenicsubtraction, i.e., the blocking of antigenic sites shared by twogenetically similar cell types. This process results in an increase inthe sensitivity of detection of novel surface antigens. The presentstudies have emphasized applications of the SEM approach, usingtransfected cells expressing known as well as unidentified cell-surfacemolecules. However, many additional situations that would be adaptableto the SEM procedure can be envisioned. For example, the SEM protocolcould be used to develop monoclonal antibodies specific for surfacechanges occurring in metastatic tumor cells. To achieve this goal,polyclonal antibodies could be generated against a primary tumor, andthese polyclonal antibodies could be used to mask surface epitopes onmetastatic tumors. This step would be performed prior to sensitizinganimals for the development of hybridomas or combinatorial phage cDNAexpression libraries specific for surface-expressed metastatic antigens.Similarly, polyclonal antibodies could be generated against normaltissue of a specific histologic type, and these polyclonal antibodiescould then be used to mask surface epitopes on a histologically similartumor derived from the same patient. The cells with masked surfaceepitopes could then be injected into animals so that they would developsensitized spleen cells for the development of hybridomas orcombinatorial phage cDNA libraries specific for tumor-associatedantigens. SEM would also appear to be ideally suited for the developmentof monclonal antibodies specific for the outer domain ofmembrane-localized growth factor receptors and cell-membrane transporterproteins. Future applications of the SEM approach could also result inthe development of monoclonal antibodies and/or the isolation ofrelevant genes involved in determining tumor cell recognition by bothnonspecific and specific immunologic effector cells, mediating atypicalmultidrug resistance, and identifying mediators of autoimmune diseases.

REFERENCES OF THE FIRST SERIES OF EXPERIMENTS

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12. Fisher, P. B., Babiss, L. E., Weinstein, I. B., et al. (1982)Analysis of type 5 adenovirus transformation with a cloned rat embryocell line (CREF), Proc. Natl. Acad. Sci. USA, 79:3527-3531.

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15. Reddy, P. G., Graham, G. M., Datta, S., et al. (1991) Effect ofrecombinant fibroblast interferon and recombinant immune interferon ongrowth and the antigenic phenotype of multidrug-resistant humanglioblastoma multiforme cells, J. Natl. Cancer Inst. 83:1307-1315.

16. Vita, J. R., Edwalds, G. M., Gorey, T., et al. (1988) Enhanced invitro growth suppression of human glioblastoma cultures treated with thecombination of recombinant fibroblast and immune interferons, AnticancerRes., 8:297-302.

17. Su, Z-z., Grunberger, D., Fisher, P. B., (1991) Suppression ofadenovirus type 5 E1A-mediated transformation and expression of thetransformed phenotype by caffeic acid phenethyl ester (CAPE), Mol.Carcinog., 4:231-242.

18. Giovanella, B. C., Stehlin, J. S., Jr., Santamaria, C., et al.(1976) Human neoplastic and normal cells in tissue culture: I. Celllines derived from malignant melanomas and normal melanocytes, J. Natl.Cancer Inst., 56:1131-1142.

19. Goldstein, N. L., Nagle, R., Villar, H., et al. (1990) Isolation andcharacterization of a human monoclonal antibody which reacts with breastand colorectal carcinoma, Anticancer Res., 10:1491-1500.

20. Duigou, G. J., Su, Z-z., Babiss, L. E., et al. (1991) Analysis ofviral and cellular gene expression during progression and suppression ofthe transformed phenotype in type 5 adenovirus-transformed rat embryocells, Oncogene, 6:1813-1824.

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22. Leon, J. A., Mesa-Tejada, R., Gutierrez, M. C., et al. (1989)Increased surface expression and shedding of tumor associated antigensby human breast carcinoma cells treated with recombinant humaninterferons or phorbol ester tumor promoters, Anticancer Res.,9:1639-1647.

23. Gottesman, M. M., Pastan, I, (1993) Biochemistry of multidrugresistance mediated by the multidrug transporter, Annu. Rev. Biochem.,62:385-427.

24. Hollingsworth, M. A., Rebellato, L. M., Moore, J. W. et al. (1986)Antigens expressed on NIH 3T3 cells following transformation with DNAfrom a human pancreatic tumor, Cancer Res., 46:2482-2487.

25. Scuderi, P., Westin, E., Clagett, J., et al. (1985) Detection ofsurface antigen in N1H 3T3 cells transfected with a human leukemiaoncogene, Med. Oncol. Tumor Pharmacother., 2:233-242.

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28. Huse, W. D., Sastry, L., Iverson, S. A., et al. (1989) Generation ofa large combinational library of the immunoglobulin repertoire in phagelambda, Science, 249:1275-1281.

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Sci. USA, 83:1453-1457.

Second Series of Experiments

Elucidating the relevant genomic changes mediating development andevolution of prostate cancer is paramount for effective diagnosis andtherapy. Using an improved DNA-acceptor cell line, CREF-Trans 6, andcotransfection techniques, with human prostatic carcinoma DNA, aputative dominant-acting nude mouse tumor-inducing oncogene, PTI-1, hasbeen identified and cloned. Differential RNA display reveals a novel 214bp DNA fragment representing a differentially expressed RNA intumor-derived transfected cells. Screening of a human prostaticcarcinoma (LNCaP) cDNA library with the novel 214 bp DNA sequenceidentifies a full-length 2.0 Kb PTI-1 cDNA. Sequence analysis indicatesthat PTI-1 is a novel gene containing a unique 630 bp 5′ sequence and a3′ sequence homologous to a truncated and mutated form of humanelongation factor-1 alpha. In vitro translation demonstrate that thePTI-1 cDNA encodes a predominant ˜46 kDa protein. Probing Northern blotswith a DNA fragment corresponding to the 5′ region of the PTI-1 geneidentifies multiple PTI-1 transcripts in RNAs from LNCaP-transfectedtumor-derived CREF-Trans 6 cells and human carcinoma cell lines derivedfrom the prostate, lung, breast and colon. In contrast, PTI-1 RNA is notpresent in human melanoma, neuroblastoma, osteosarcoma, normalcerebellum or glioblastoma multiforme cell lines. Using a pair ofprimers recognizing a 279 bp region within the unique 630 bp 5′ PTI-1sequence, RT-PCR detects PTI-1 expression in patient-derived prostatecarcinomas, but not in normal prostate or benign prostatic hypertrophy(BPH). In contrast, RT-PCR detects prostate-specific antigen (PSA)expression in all prostates tissue specimens. These results indicatethat PTI-1 is a novel putative oncogene that may contribute to carcinomadevelopment in human prostate and other tissues. The approaches used,rapid expression cloning with the CREF-Trans 6 system and thedifferential RNA display strategy, should prove widely applicable foridentifying and cloning additional novel human oncogenes.

The American Cancer Society estimates that 200,000 American men willhave been diagnosed with prostate cancer in 1994 and 38,000 afflictedmen will have died of this disease (1). The current methods fordetecting early prostate cancer are limited in both their sensitivityand specificity (2). These include physical examination that mighteasily miss small or centrally located tumors, serum prostate-specificantigen (PSA) determination that is not specific to malignant prostatedisease, and tissue biopsy in which sampling error may lead to erroneousbenign diagnosis (3, 4). Predictors and early detection of therapeuticrelapse such as monitoring of PSA levels, ultrasound and bone scans arealso unsatisfactory, as these require fairly bulky tumor regrowth beforediscovery (5, 6). Using current approaches a high percentage, 40 to 50%,of patients considered to have clinically localized disease actuallycontain understaged diseases subsequent to radical surgery (7, 8).Surgical intervention is not considered the appropriate treatmentprotocol for patients with progressive disease. These findings emphasizethe need for improved diagnostic and therapeutic approaches foridentifying prostate carcinomas and for predicting clinicalaggressiveness.

A primary objective of investigators studying cancer etiology is theidentification of gene(s) within tumor cells with oncogenic potential. Aprocedure to achieve this goal involves the transfer of high molecularweight (HMW) DNA from established tumor cell lines or primary tumorsinto appropriate acceptor cell lines by DNA-transfection (9). Targetcells are then examined for morphological transformation, i.e., focusformation. A modification of this approach involves cotransfection oftarget cells with HMW DNA plus a selectable antibiotic resistance gene,such as pSV2neo, selection for antibiotic resistance and then injectionof pooled antibiotic resistant cells into nude mice to identify clonesof cells with tumorigenic potential (10). The majority of studies usingthese approaches have relied on the immortal murine cell line NIH-3T3(9, 10). Unfortunately, NIH-3T3 cells generally prove unsuccessful inidentifying novel dominant-acting oncogenes from human tumor cell linesor clinical samples and even when successful, subsequent cloningindicates genetic elements not relevant to the majority of humancancers. These studies accentuate the need for better techniques toidentify dominant-acting human cancer genes and for more suitable targetcell lines to detect novel tumor-inducing oncogenes.

Recent studies using the cotransfection/nude mouse tumor assay with HMWDNA from a human prostatic carcinoma cell line, LNCaP (11), and a newDNA-acceptor cell line, CREF-Trans 6 (12), indicate the presence of adominant-acting tumor-inducing gene (12). Applicants have presentlycloned and characterized a novel gene, prostate carcinoma tumor inducinggene 1 (PTI-1), using the differential RNA display (DD) technology (13),library screening strategies (14, 15) and the RACE procedure (14). Thefull-length PTI-1 cDNA consists of 2,123 nucleotides and contains anovel 630 nt region sharing sequence homology with bacterial ribosomal23S RNA fused to a sequence that is a truncated and mutated form ofhuman elongation factor-1 alpha (EF-1α). LNCaP-transfected tumor-derivedCREF-Trans 6 cells as well as human prostate carcinoma cell lines andpatient-derived prostate carcinomas express PTI-1. In contrast, PTI-1RNA is not evident using RT-PCR in normal prostate or benign prostatichypertrophy (BPH) tissue samples. PTI-1 expression occurs in additionalhuman carcinomas, including breast, colon and lung, but not in normalcerebellum, glioblastoma multiforme, melanoma, neuroblastoma orosteosarcoma cell lines. These observations indicate that PTI-1 is anovel genetic element displaying expression in specific human carcinomasand implicates mutagenic changes in EF-1α as a contributor to thecarcinogenic process.

Materials and Methods

Cell lines. The LNCaP cell line was derived from metastatic depositsfrom a patient with advanced prostate cancer (11) and was provided byDr. Steven Harris (W. Alton Jones Cell Science Center, N.Y.) CREF-Trans6 and LNCaP DNA-transfected nude mouse tumor-derived CREF-Trans 6 cells,CREF-Trans 6:4 NMT, were isolated as described previously (12). Thehormone independent prostatic carcinoma cell line DU-145, theendometrial carcinoma cell line HTB-113, the small cell lung carcinomacell line NCI-H69 and the human neuroblastoma cell line IMR-32 wereobtained from the American Type Culture Collection. The nasopharyngealcarcinoma cell line KB 3-1 was provided by Dr. Michael M. Gottesman(NCI, MD). The human breast carcinoma cell line MCF 7 and the humancolon carcinoma cell lines WiDr, HT 29, SW480 and LS174T were suppliedby Dr. John W, Greiner (NCI, MD). The human breast carcinoma cell lineT47D was provided by Dr. Ricardo Mesa-Tejada (MetPath Inc., NJ). Anormal human cerebellum cell line, a human glioblastoma multiforme cellline GBM-18 and a human neuroblastoma cell line NB-11 were establishedin the applicants' laboratory (17-19). H0-1 human melanoma cells wereobtained from Dr. Beppino Giovanella (Stehlin Foundation, TX). C8161metastatic human melanoma cells were supplied by Dr. Danny R. Welch(Hershey Medical Center, PA). The human osteosarcoma cell line Saos-2was provided by Dr. C. S. Hamish Young (Columbia Univ., NY). Conditionsfor growing the various cell types were as described previously (11, 12,17-19).

RNA preparation, differential RNA display (DD) and RT-PCR. Totalcytoplasmic RNA was isolated from logarithmically growing cell culturesas previously described (14, 15). Tissue samples from normal prostatesand patients with prostatic carcinomas or BPH were frozen in liquidnitrogen and RNA was isolated using the TRIzol reagent as described byGibcoBRL (MD). Tissue samples were supplied by the Cooperative HumanTumor Network (CHTN). Samples of normal prostate were obtained fromautopsies of males <40 years of age. All tissues were histologicallyconfirmed as normal, BPH or carcinoma of the prostate. The DD procedurewas performed essentially as described by Liang and Pardee (13). Two μgof mRNAs from CREF-Trans 6 and CREF-Trans 6:4 NMT cells were reversetranscribed with 300 units of MMLV reverse transcriptase (BRL) in thepresence of 2.5 μM of primer T12GC (5′-TTTTTTTTTTTTGC-3′) and 20 μM dNTPmix (BRL) for 60 min at 35° C. Two μg of the cDNA was PCR-amplified inthe presence of 2 μM T12GC and 2 μM of a 5′-primer JB-24(5′-ACCGACGTCGACTATCCATGAACA-5′). Samples were resolved in parallellanes on a 5% denaturing sequencing gel and differentially expressedbands were removed from the gel and electroeluted in 0.2× TBE solution.The same pair of primers were used for PCR amplification of thedifferentially expressed sequences followed by TA cloning kit(Invitrogen). Plasmids containing inserts of the predicted size weresequenced by the Sanger method (Sequenase kit, version 2.0, USB) or theinserts were isolated and used to probe Northern blots (14-16). RT-PCRusing appropriate primers was performed as described previously (16).

cDNA library construction, screening and sequencing. A cDNA library ofLNCaP mRNA was constructed in the Uni-ZAP XR vector (Stratagene) andscreened as previously described (14). A 1.8 kb PTI-1 DNA fragment wasobtained by RT/PCR amplification of LNCaP cDNA with a 20 mer(5′-AACTAAGTGGAGGACCGAAC-340 ) within the 214 bp DNA obtained by DD.Inserts from the plasmids containing the largest PTI-1 inserts wereexcised by digestion with the restriction enzymes XhoI and EcoRI andtested by Northern blotting with appropriate RNA samples and sequencedusing the Sanger method with an Applied Biosystems (Model 373A, Version1.2.1) sequencer and oligonucleotides synthesized from both ends of thegene inserts.

RACE procedure. To identify the 5′-extended region of PTI-1, a 22 baseoligomer (I) (5′-CCTTGCATATTAACATAACTCG-3′) and a 19 base oligomer (II)(5′-AAGTCGCCCTATTCAGACT-3′), antisense direction of the sequences262-283 bp and 317-336 bp, respectively, were synthesized. The RACEprotocol was performed using the 5′ RACE system (GibcoBRL, MD) aspreviously described (14).

In vitro translation of PTI-1 encoded proteins. PTI-1 was linearized bydigestion with XhoI and used as a template to synthesize mRNA using themCAP mRNA capping kit (Stratagene). In vitro translation of PTI-1 wasperformed using a rabbit reticulocyte lysate translation kit withconditions as described by GibcoBRL (MD).

Experimental Results

Identification and properties of PTI-1. The rapid expression cloningsystem identified a potential S oncogenic element in LNCaP cells (12).To identify genes displaying differential expression in CREF-Trans 6cells and nude mouse tumor-derived LNCaP-transfected CREF-Trans 6 cells,CREF-Trans 6:4 NMT, the DD approach developed by Liang and Pardee (13)was used. This protocol permits identification of differentiallyexpressed cDNAs based on size as opposed to nucleotide composition orfunction. A problem often encountered using DD is the identification ofamplified sequences not displaying differential expression when testedusing appropriate RNA samples and Northern blotting (12). An example ofthis type of artifact is seen in FIG. 6, i.e., the band present inCREF-Trans 6:4 NMT directly below the arrow. The frequency of falsesignals can be significantly reduced by using subtraction hybridizationprior to PCR amplification and DD (data not shown). Using DD, a 214 bpDNA fragment (PTI-1) was identified in the LNCaP-transfected nude mousetumor-derived CREF-Trans 6 cell line, CREF-Trans 6:4 NMT, that was notpresent in parental CREF-Trans 6 cells (FIG. 6, arrow). The PTI-1fragment was isolated, cloned, sequenced and used to probe Northernblots containing RNAs from CREF-Trans 6, CREF-Trans 6:4 NMT and LNCaPcells (FIG. 7). The 214 bp PTI-1 DNA fragment is a novel sequence andhybridizes to several RNAs present in CREF-Trans 6:4 NMT, LNCaP and thehormone independent prostate carcinoma cell line DU-145 (FIG. 7).

The complete sequence of PTI-1 is presented in FIG. 8.

PTI-1 consists of 2,123 bp, the 5′-flanking region (1 to 215 bp) wasobtained by RACE 5′-extension and the remainder of the gene (216 to2,123 bp) was determined by direct sequencing. Primer extension analysisand RT-PCR of LNCaP mRNA confirm that PTI-1 is a full-length cDNA (datanot shown). The 3′ region of PTI-1 extending from 630 to 2,123 bpdisplays 97% homology to a truncated human EF-1α gene. The 5′ region ofPTI-1 displays no homology to eucaryotic genes, but instead is ˜85%homologous to procaryotic 23S ribosomal RNA gene from Mycoplasmahyopneumoniae. This region of PTI-1 contains the 214 bp DNA marker(core) sequence obtained using DD (FIG. 6). The unique 5′ region alsocontains a large number of stop codons (TAA, TGA and TAG sequences)(FIG. 8). These observations suggest that PTI-1 is a fusion geneconsisting of two regions: a 5′ unique 630 bp region and a 3′ truncatedand mutated EF-1α gene.

PTI-1 contains an open-reading frame from bp 621 to 1,814 with a stopcodon after the last amino acid K and encodes a protein of 398 aa (FIG.8). A comparison of the amino acid sequence of PTI-1 (1 to 398 aa) and apartial human EF-1α (aa 1 to 462) is presented in FIG. 8. PTI-1 and thetruncated human EF-1α share 98.4% similarity and 97.7% identity. PTI-1contains the same carboxyl terminus as human EF-1α. The N-terminus ofPTI-1 is different from human EF-1α and consists of a deletion of 67 aanormally found in human EF-1α and an insertion of 3 unique amino acids(MQS) in PTI-1 that differs from the original N-terminus (MGK) of humanEF-1α. In addition, 6 in frame amino acid changes are present in PTI-1(FIG. 8). The loss of 67 amino acids in the N-terminus plus changes inspecific amino acids, from positive charged to non-positive chargedamino acids and from hydroxyl group-containing to non-hydroxylgroup-containing amino acids, can be anticipated to impact on the threedimensional structure and functionality of this mutant EF-1α protein. Onthe basis of sequence analysis, PTI-1 should encode a protein of 43.8kDa. To confirm this prediction, in vitro translation analyses ofproteins encoded by the PTI-1 cDNA were determined (FIG. 9). Apredominant protein present after in-vitro translation of PTI-1 has an Mof ˜46 kDa. This value is larger than predicted and may result becauseof protein modification, i.e., phosphorylation, in the rabbitreticulocyte lysate system. Four additional minor proteins (M 41 to 30.5kDa) are also present after in vitro translation. These proteinsprobably result from initiation of protein synthesis at start codons(ATG) downstream of the first start codon in PTI-1 (FIG. 8).

Expression of PTI-1 in RNA samples from patient-derived tissues and celllines. An important question is whether PTI-1 expression occurs inprostatic carcinomas in vivo. For this analysis, RNA was isolated fromquick-frozen prostate samples from patients obtained during operationsand confirmed as prostatic carcinomas or BPH histologically. RNAs werealso extracted from normal prostates obtained at autopsy from men lessthan 40 years of age and histologically normal. Using RT-PCR withprimers (A and L) (FIG. 8) synthesized from the unique 630 bp 5′ PTI-1sequence, expression is apparent in seven of eight human prostaticcarcinomas (FIG. 10 and data not shown). In contrast, PTI-1 is notexpressed in four normal prostates or three BPH patient samples (FIG.10). In contrast, LNCaP and all prostate samples, including normal, BPHand carcinoma, are positive for PSA expression, whereas CREF-Trans 6,CREF-Trans 6:4 NMT and DU-145 do not express PSA (FIG. 10). All sampleswere positive for GAPDH expression (FIG. 10). These results indicatethat PTI-1 is expressed in human prostate carcinomas, but not in normalprostates or BPH.

To determine the pattern of PTI-1 expression in additional cell types,RNAs from various cell lines were analyzed by Northern blotting usingPTI-1 and GAPDH as probes (FIG. 7). In addition to being expressed inCREF-Trans 6:4 NMT, LNCaP and DU-145, PTI-1 expression is evident inother human carcinomas, including NCI-H69 (small cell lung), T47D(breast), and SW480 and LS174T (colon). In contrast, PTI-1 expression isnot detected in HTB-113 (endometrial adenocarcinoma), KB 3-1(nasopharyngeal carcinoma), MCF 7 (breast carcinoma), WiDr and HT 29(colon carcinoma), normal cerebellum, GBM-18 (glioblastoma multiforme),H0-1 and C8161 (melanoma), NB-11 and IMR-32 (neuroblastoma) or Saos-2(osteosarcoma) cells. These observations indicate that PTI-1 expressionis not restricted to human prostate carcinoma, but also occurs in ˜50%of the human carcinomas analyzed.

Experimental Discussion

Cancer is a progressive disease in which tumor cells manifest continuousgenetic changes that correlate with increasing frequencies ofchromosomal abnormalities and mutations (rev. 20-22). Recent studiessuggest that mutations in genes involved in maintaining genomicstability, including DNA repair, mismatch repair, DNA replication andchromosomal segregation, may result in acquisition of a mutatorphenotype by cancer cells predisposing them to further mutationsresulting in tumor progression (rev. 21). In leukemias as well asspecific solid tumors, improved cytogenetic techniques and molecularapproaches indicate that specific translocations result in theactivation of proto-oncogene products and the creation of tumor-specificfusion proteins (22). A common observation is that both types of noveloncogenic elements are often transcription factors suggesting thatalterations in transcriptional control may directly contribute to cancerdevelopment and evolution (22, 23). Modifications in the translationalmachinery of cells, including changes in both eucaryotic initiationfactors and elongation factors, can also result in susceptibility totransformation and the acquisition of transformed and oncogenicproperties in specific target cells (rev. 24, 25). For example,overexpression of a normally rate-limiting protein initiation factor,eIF-4E, can cooperate with both the v-myc and adenovirus E1A gene ininducing transformation of primary rodent fibroblasts (26), inducetumorigenic transformation in both NIH 3T3 and Rat 2 cells (27) andinduce in combination with Max both a tumorigenic and metastaticphenotype in Chinese hamster ovary (CHO) cells (28) Enhanced expressionof elongation factor-1a (EF-1α), a nucleotide exchange protein thatbinds GTP and aminoacyl-tRNA and results in codon-dependent placement ofthis aminoacyl-tRNA at the A site of the ribosome (24, 25), conferssusceptibility to carcinogen- and ultraviolet light-inducedtransformation to mouse and Syrian hamster cell lines (29). Elevatedlevels of wild-type EF-1α also occur in tumors of the pancreas, colon,breast, lung and stomach relative to normal tissue (30). Moreover,enhanced expression of EF-1γ, a nucleotide exchange protein thatmediates transport of aminoacyl tRNAs to 80S ribosomes during RNAtranslation, is found in a high proportion of pancreatic tumors (78%),colorectal tumors (86%) and colorectal adenomas (56%) relative tonormal-appearing adjacent tissue (31-33). These findings indicate thatalterations in both gene transcription and protein synthetic processescontribute to oncogenesis.

The present study implicates a novel gene, PTI-1, that contains a uniquesequence linked to a truncated and mutated EF-1α gene, in oncogenictransformation and prostate carcinoma development. PTI-1 is expressed inLNCaP-transfected tumor-derived CREF-Trans 6 cells, human prostaticcarcinoma cell lines and patient-derived carcinomas, whereas expressionis not detected in normal prostate or BPH tissues. PTI-1 RNA is alsofound in additional human carcinomas, of the breast, lung and colon.These results indicate that PTI-1 expression may be a common alterationin human carcinomas. The direct cloning of PTI-1 from an LNCaP cDNAlibrary indicates that this novel gene is originally present in thisprostatic carcinoma cell line and does not develop as a consequence ofmutation resulting during transfection into CREF-Trans 6 cells orselection for tumor-formation in nude mice.

EF-1α is analogous to bacterial elongation factor-Tu (EF-Tu), bothmembers of the GTPase superfamily of proteins (rev. 34-36). A primaryfunction of EF-Tu/EF-1α is the process of kinetic proofreading thatresults in appropriate codon-anticodon binding interactions (36).Mutations in specific regions of EF-Tu result in altered biologicalfunction, including a dominant negative inhibition of protein synthesisby mutational replacement of Lys 136 by glutamate or glutamine in theG-4 GTPase region that interacts with guanine nucleotide releaseproteins (GNRPs) (37). EF-Tu mutants in Escherichia coli and Salmonellaexhibit increases in missense error rates (38, 39). Mutations in EF-1αcan directly affect the frequency of frameshifting and amino acidmisincorporations in Saccharomyces cerevisiae (40). Single amino acidsubstitutions in EF-1α alter the selection and/or proofreading of thecodon-anticodon match (40). Moreover, altering the level of EF-1α inSaccharomyces cerevisiae directly affects suppression of nonsensemutations further indicating a critical involvement in translationalfidelity (41). In this context, the mutated EF-1α protein encoded byPTI-1 could modify normal EF-1α function resulting in decreased proteintranslational fidelity and an inability to suppress specific mutationsin carcinomas. If this “translational infidelity” hypothesis is correct,PTI-1 may represent a mutated “1genomic stability” gene (21) and animportant contributor to the mutator phenotype of cancer cells and tumorprogression.

An important early event in carcinogenesis may involve mutations thatconfer immortality or an enhanced cellular life span (20, 21). Duringcellular senescence the levels and catalytic activity of EF-1α decrease(42). Forced expression of EF-1α in Drosophila melanogaster extendslife-span in comparison with control flies (43). The reduction inproliferative capacity associated with senescence correlates with areduced capacity for mitosis. In this respect, the recent demonstrationthat EF-1α may be an important element in mitotic spindle formation (44)may be relevant. As demonstrated in this report, the EF-1α sequence inPTI-1 contains a deletion of 67 amino acids and six point mutations incomparison with wild-type human EF-1α (FIG. 8). Although the relevanceof these alterations to EF-1α activity are unknown, it is possible thatthis gene undergoes a series of step-wise mutations during prostatecancer development. If this hypothesis is correct, changes in thestructure of the PTI-1 gene could represent a genetic marker forprostatic carcinoma development and progression. Studies are currentlyin progress to test these hypotheses and to determine if expression ofPTI-1 and/or genetically modified EF-1α genes in CREF-Trans 6 cellsresults in acquisition of oncogenic potential.

A previous limitation preventing the identification and cloning of noveloncogenes was the absence of a sensitive transfectable indicator cellline. This problem has been ameliorated with the identification of theCREF-Trans 6 clone (12). Using rapid expression cloning with theCREF-Trans 6 acceptor cell line and the DD technology, the novelputative oncogene PTI-1 displaying expression in human prostate, breast,lung and colon carcinomas has been identified and cloned. In comparativestudies using NIH-3T3 cells, cotransfection of high molecular weight DNAfrom LNCaP cells and antibiotic resistance plasmid (pSV2neo) DNA did notresult in tumors following injection of G418-resistant cells into nudemice (12). Rapid expression cloning with CREF-Trans 6 also results inthe transfer of tumor-inducing oncogenes from a human breast carcinoma,a glioblastoma multiforme and a small cell lung carcinoma cell line andfrom a patient-derived metastatic colon carcinoma lesion (data notshown). Although the identifications of the dominant-acting geneticelements present in these human tumor DNA-transfected CREF-Trans 6clones are not known, these exciting preliminary results suggest thatthis new acceptor cell line could prove useful for identifying andcloning potentially novel human oncogenes involved in the development ofdiverse human cancers.

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Third Series of Experiments

The American Cancer Society estimates that 200,000 American men willhave been diagnosed with prostate cancer in 1994 and 38,000 afflictedmen will have died of this disease. The current methods for detectingearly prostate cancer are limited in both their sensitivity andspecificity. These include physical examination that might easily misssmall or centrally located tumors, serum prostate-specific antigen (PSA)determination that is not specific to malignant prostate disease, andtissue biopsy in which sampling error may lead to erroneous benigndiagnosis. Predictors and early detection of therapeutic relapse such asmonitoring of PSA levels, ultrasound and bone scans are alsounsatisfactory, as these require fairly bulky tumor regrowth beforediscovery.

Despite intensive scientific effort, the relevant genomic changes thatmediate the development and evolution of prostate cancer remain to bedefined. In addition, biochemical and molecular markers correlating withpotential aggressiveness of a specific prostate carcinoma and theappropriate therapy that will effectively prevent disease progressionare not currently available. It is now well established that many formsof cancer are the result of complex multifactor interactions andcarcinogenesis is a multistep process. Genetic factors contributing tocarcinogenesis include dominant acting oncogenes that promote the cancerphenotype and tumor suppressor genes that function as negativeinhibitors of the cancer process. Our broad goals are to define prostatecancer in molecular terms and use this information to design betterdiagnostic tools and therapies for this malignancy. To achieve theseobjectives it will be necessary to identify and characterize genes thatcan both induce and inhibit this disease process. Once appropriategenetic mediators of human prostate cancer are identified thisinformation will prove valuable for developing more effective diagnostictools and ultimately for generating improved gene-based andimmunologically-based therapies for this pervasive cancer.

The Rapid Expression Cloning (RExCS) System

A primary objective of investigators interested in the etiology of humancancer is the identification of gene(s) within tumor cells withoncogenic potential. One procedure used to achieve this goal involvesthe transfer of high molecular weight (HMW) DNA isolated fromestablished tumor cell lines or primary tumors into appropriate celllines by calcium-mediated DNA-transfection, lipofection, electroporationor other approaches. Target cells are then examined for signs ofmorphological transformation, i.e., focus formation. A modification ofthis approach involves cotransfection of target cells with HMW DNA plusa selectable antibiotic resistance gene, such as pSV2neo, selection forantibiotic resistance and then injection of pooled antibiotic resistantcells into nude mice to identify clones of cells with tumorigenicpotential. The majority of studies using these approaches have relied onthe immortal murine cell line NIH-3T3. Unfortunately, NIH-3T3 cells havegenerally not proven successful in identifying novel dominant-actingoncogenes from human tumor lines or clinical samples and even whensuccessful, subsequent cloning of the transforming gene has revealedgenetic elements not relevant to most human cancers. These findingsemphasize the need for improved techniques to identify dominant-actinghuman cancer genes and the identification of more suitable target celllines that can express novel tumor-inducing human oncogenes.

To identify dominant acting oncogenes in human prostate carcinoma cellsapplicants have used 2 approaches, both utilizing DNA cotransfectiontechniques with a new DNA-acceptor cell line, CREF-Trans 6, and tumorformation in nude mice as an endpoint. Cotransfection of CREF-Trans 6with HMW DNA from the human prostate carcinoma cell line LNCaP andpSV2neo DNA, selection for G418 resistance and injection into nude miceresulted in tumor formation. In contrast, no transformed foci wereapparent in similarly transfected CREF-Trans 6 cells maintained only inmonolayer culture. No dominant acting focus forming or tumor inducingoncogene was detected in NIH-3T3 cells cotransfected with LNCaP andpSV2neo DNA. Both primary and secondary nude mouse tumor-derivedCREF-Trans 6 cells contain human repetitive (Alu) sequences that are notpresent in untransfected CREF-Trans 6 cells. A common Alu fragment ispresent in Southern blots in both primary and secondary tumor derivedCREF-Trans 6 cells. Tumor-derived CREF-Trans 6 cells also containadditional Alu sequences of different apparent molecular sizes. Thisdata provided initial supportive evidence that a human gene(s)potentially capable of inducing a tumorigenic phenotype innontumorigenic CREF-Trans 6 cells had been transferred from the humanprostate carcinoma cell line LNCaP. Using both molecular andimmunological approaches tumor-derived CREF-Trans 6 cells have been usedto: (a) identify and clone novel genes, termed prostate tumor inducinggenes (PTI-1, PTI-2, FIG. 13 and PTI-3, FIG. 14), potentially involvedin the etiology of human prostate carcinoma; and (b) produce monoclonalantibodies reacting with the surface of human prostate carcinoma cellsand the cloning of a cDNA encoding a novel tumor associated antigen,termed prostate carcinoma tumor antigen gene (PCTA-1, FIG. 15).

Other Applications of RExCS: Cotransfection of CREF-Trans 6 with pSV2neoDNA and high molecular weight DNA from a human glioblastoma multiformecell line (GBM-18), a human breast carcinoma cell line (T47D) and ahuman small cell lung carcinoma cell line (NCI-H69) results in tumorformation in nude mice. Similarly, cotransfection of CREF-Trans 6 withpSV2neo DNA and high molecular weight DNA from a patient-derivedmetastatic colorectal carcinoma results in tumor formation in nude mice.Tumor-derived cell lines have been isolated and can now be used: toclone the transforming genetic elements mediating the tumorigenicphenotype; and with the SEM procedure to develop potentially novel MAbsreacting with TAAs expressed by specific human cancers.

Prostate Tumor Inducing Gene-1 (PTI-1): PTI-1 was initially identifiedin LNCaP DNA transfected tumor-derived CREF-Trans 6 cells using anapproach termed RNA differential display (DD). DD permits theidentification and cloning of differentially expressed mRNAs encoded byclosely related cell types. The basic DD approach involves a series ofinterrelated steps, including: (a) isolating mRNA from two closelyrelated cell types; (b) producing reverse transcribed-PCR (RT-PCR)products using a primer that anchors the PCR products to the 3′ end ofthe mRNA and 5′ primers containing arbitrary oligonucleotides of varioussizes; (c) running RT-PCR products from both cell types in adjacentlanes of a sequencing gel; (d) cutting differentially expressed bandsout of the sequencing gel, eluting the PCR product and PCRamplification; (e) testing for expression of the PCR product usingNorthern blots containing relevant RNA samples; and (f) sequencingappropriately expressed sequences to determine identity with previouslyreported genes. Improvements in DD include the use of a subtractionhybridization step prior to performing RT-PCR. This technicalimprovement to DD results in a dramatic reduction in the number of falsepositives, that can exceed 40% using standard DD.

The DD cloning strategy has now been successfully used withuntransfected CREF-Trans 6 cells and an LNCaP DNA transfected nude mousetumor derived CREF-Trans 6 clone, CREF-Trans 6: 4 NMT to identify PTI-1(PTI-2 and PTI-3- to be described below). An anchored oligo-dT primerconsisting of 12 Ts plus two additional 3′ bases, that providesspecificity, was used to anneal the beginning of a subpopulation of thepoly(A) tails of the mRNAs for reverse transcription. A set of arbitraryprimers was used as a 5′-primer for PCR amplification of the cDNAsgenerated by reverse transcription from the mRNAs. These amplified cDNAfragments were then separated by size to a maximum of 500 bp on adenaturing polyacrylamide gel. A differentially expressed band of 214 bpthat was present in CREF-Trans 6: 4 NMT but not in CREF-Trans 6 was cutout of the gel, electroeluted in TBE and reamplified by PCR using thesame primers as used for DD. The purified 214 bp DNA fragment from the1% agarose gel was then cloned into the PCR™II vector and transformed inOneShot™ competent cells. This sequence referred to as 214 bp PTI-1, wastested for expression using Northern blotting analysis. PTI-1 wasexpressed in LNCaP and CREF-Trans 6: 4 NMT, but not in CREF-Trans 6,human breast carcinoma cells (MCF7), human glioblastoma multiforme(GBM-18) or human melanoma cells (H0-1 and C8161). Sequence analysis ofthe 214 bp PTI-1 DNA fragment using the Sanger sequencing procedureindicates no homology to previously reported genes deposited in variousgene banks (GenBank (R), Brookhaven Protein Data Bank, EMBL DataLibrary). In order to identify the 5′- and 3′-flanking regions of the214 bp DNA, rapid amplification of cDNA ends (RACE) was performed. TheRACE approach is a procedure for amplification of nucleic acid sequencesfrom a mRNA template between a defined internal site and an unknownsequence representing either the 3′ or 5′ end of the mRNA. Using RACEand primers designed from the 214 bp PTI-1 sequence, a 1.8 Kb PTI-1 DNAfragment was generated by PCR. Northern analysis using the 1.8 Kb PTI-1DNA fragment produced the same reactivity pattern as observed with the214 bp PTI-1 DNA fragment. Approximately 600 bp of the 5′ region of thePTI-1 DNA fragment was sequenced and found to display no homology toreported eucaryotic gene sequences, but rather ˜85% homology to 23Sribosomal RNA from Mycoplasma hyopneumoniae). This 1.8 Kb PTI DNA wascalled PTI-3 and will be described later. This 1.8 Kb PTI DNA was laterused to screen an LNCaP cDNA library constructed in the Uni-ZAP XRvector. Two clones were identified. One is clone 18, which is calledPTI-1; another, clone 8, is called PTI-2 and will be described later.

The PTI-1 gene is composed of two parts; one is 5′-RACE extended region(1-215 bp) and another clone 18 part. Clone 18 (PTI-1) contains 1937 bp(29 bp+1908 bp) insert in pBluescript vector. The experimentdemonstrates that the 29 bp at the 5′-end comes from wrong reversetranscription because of lower temperature and secondary structure ofRNA, so that this 29 bp sequence was replaced by the right sequenceobtained by RACE method and did not show in the complete sequence ofPTI-1. The sequence from 30-1937 bp (1907 bp) of clone 18 was shown asthe sequence 216 bp-2123 bp (1907 bp) in FIG. 3. The plasmid deposit isclone 18 (PTI-1). A pair of oligonucleotides with sequences in the 5′region of the 1.8 Kb PTI-1 DNA was synthesized and RT-PCR was performedusing mRNA isolated from both cell lines and human tissue samples. Thisanalysis indicates that PTI-1 is expressed in CREF-Trans 6: 4 NMT,LNCaP, DU-145 (a hormone independent human prostate carcinoma cellline), 7 of 8 patient-derived prostate carcinomas. In contrast, PTI-1 isnot expressed in CREF-Trans 6, H0-1, MCF-7 or tissue from normal humanprostates or benign prostatic hypertrophy (BPH). These studies indicatethat PTI-1 is a novel human oncogene that may be a mediator of or thatis associated with transformation and tumorigenesis in human prostatecarcinoma cells.

To identify a full-length PTI-1 cDNA, an LNCaP cDNA library wasconstructed in the Uni-ZAP XR vector. Screening the LNCaP library usingthe 1.8 Kb PTI-1 DNA probe resulted in the identification of an ˜2.0 KbPTI-1 cDNA from this library. Two approaches indicate that this PTI-1cDNA is a full-length cDNA. One approach uses primer extension analysisof the ˜2.0 Kb cDNA and the second approach involves in vitrotranslation of in vitro RNA transcribed from the ˜2.0 Kb cDNA. Using areticulocyte translation system, the transcribed RNA from the ˜2.0 KbPTI-1 cDNA generates several protein products with a predominant proteinof approximately 46 kDa. Complete sequence analysis and comparison withexisting DNA data bases of the ˜2.0 Kb PTI-1 cDNA isolated from theLNCaP cDNA library indicates that PTI-1 is a novel fusion gene. PTI-1consists of a unique 630 bp 5′ sequence and a 3′ sequence homologous toa truncated and mutated form of human elongation factor-1 alpha. Afull-description of PTI-1 with sequence and specific properties can befound in our PNAS paper.

PTI-1: Using primer sequences for bases present in the unique 630 bp 5′region of PTI-1 (A and L) and primer sequences corresponding to theelongation factor-1 alpha region of PTI-1 and RT-PCR approaches, thefollowing additional information is currently available relative toPTI-1: (A) Tissue distribution studies (using tissue poly A′ mRNA blotsfrom Clontech) have been performed using the A and L primers and aregion corresponding to the elongation factor-1 alpha homologous regionof PTI-1 as probes. The unique region of PTI-1 is only expressed inskeletal muscle and colon tissue, whereas the elongation factor-1 alphahybridizes with an mRNA present in all of the tissue samples. Theseinclude, spleen, thymus, prostate, testis, ovary, small intestine,colon, peripheral blood leukocyte, heart, brain, placenta, lung, liver,skeletal muscle, kidney and pancreas. These studies reinforce ourprevious observations that the unique region of PTI-1 is not expressedin normal human prostate. (B) Expression of PTI-1 (A and L primers) isreduced in LNCaP cells treated with: a phorbol ester tumor promoter(12-0-tetradecanoyl-phorbol-13-acetate (TPA), that induces apoptosis inLNCaP cells; suramin; epidermal growth factor; transforming growthfactor-alpha; or the synthetic androgen R1881. Using primers forprostate specific antigen (PSA) reductions in PTI-1 mRNA levels usingthe same agents are also apparent in LNCaP cells. These results suggestthat similar changes inducing downregulation of PSA expression can alsodecrease PTI-1 expression in human prostate carcinoma cells. (C)Expression is apparent in human promyelocytic leukemia (HL-60) and anadditional leukemic cell line K562. When induced to differentiate byTPA, PTI-1 expression decreases and is no longer apparent by 3 hrposttreatment in HL-60 cells. This change in mRNA levels after TPAtreatment suggests that decreased expression of PTI-1 may be modulatedas a function of growth arrest and terminal differentiation in HL-60cells; (D) Expression is apparent in CREF cells transformed by diverseacting oncogenes, including wild-type 5 adenovirus (Ad5), mutant type 5adenovirus (H5hr1), Ha-ras oncogene, v-src, human papilloma virus type18 (HPV-18) and HPV-51. Using a dexamethasone (DEX) inducible Ad5 E1Atransforming gene under the transcriptional control of a mouse mammarytumor virus promoter, expression of PTI-1 is only seen in the presenceof DEX. Under these culturing conditions, DEX also results in E1Aexpression and transformation. These data indicate that induction ofPTI-1 directly correlates with transformation induced by mechanisticallydifferent oncogenes. (Figure of Northern blot; FIG. 11).

Uses for PTI-1: The unique region of PTI-1 can be used to: (A) Produceprimers for RT-PCR that will distinguish between prostate carcinoma andnormal or BPH tissue samples (diagnostic applications); (B) Permitdevelopment of a blood test to identify prostate carcinoma cells thathave metastatic potential and that have escaped from the prostate(diagnostic applications); (C) Permit identification of additionalcarcinomas, i.e., breast, colon and lung, in the blood stream that haveresulted from metastatic carcinoma spread (diagnostic applications); and(D) Develop an antisense vector and/or ribozyme approach to inhibitexpression and induce growth arrest and/or apoptosis in prostatecarcinoma cells, and perhaps other carcinoma and leukemic cells(therapeutic applications).

The mutated elongation factor i-alpha region of PTI-1 may prove usefulto: (A) Identify genetic changes in cells predisposing to carcinomadevelopment and progression (diagnostic applications); (B) Identifypoint mutations and or deleted regions of this gene that could proveuseful for predicting carcinoma development and progression (diagnosticapplications); and (C) Develop antisense and ribozyme strategies toinhibit expression of the mutant form of elongation factor 1-alpharesulting in suppression of carcinoma growth (therapeutic applications).

PTI-1 and Prostate Tumor Inducing Gene-2 (PTI-2): A cDNA library wasprepared from LNCaP cells in the Uni-ZAP XR vector (Stratagene). TheLNCaP cDNA library was screened with a ³²P-labeled 1.8 Kb DNA (PTI-3)containing the 214 bp DNA obtained by the differential displayprocedure. The contents of ten plates of 150 mm×15 mm (containing ˜2×10⁴plaques/plate) were transferred in duplicate to nylon membranes.Hybridization was performed using the following conditions: 5% dextransulfate, 45% deionized formamide, 4×SSC, 1 mM phosphate buffer (pH 7.5),0.5% SDS, 5% Denhardt's reagent at 42° C. in a Hybridization IncubatorModel 400 (Robbin Scientific); washing at 55° C. for 60 min in asolution of 0.250% SDS and 1×SSC. Positive plaques obtained in the firstround were screened in duplicate for a second round and then with invivo excision produced plasmids containing gene inserts in thepBluescript vector. Plasmids containing the longest inserts wereidentified by Southern blotting and probing with the 1.8 Kb (PTI-3) DNAprobe. Two clones were identified using this approach: clone 8 (PTI-2)and clone 18 (partial sequence of PTI-1).

The full sequence of PTI-1 contains 2,123 bp, the 5′-flanking (1-215 bp)was obtained by RACE 5′-extension (GIBCO-BRL), the remaining 216-2,123bp was obtained by sequencing clone 18. The RACE 5′ extension wasperformed with two oligonucleotides, both located within the 5′-end ofclone 18. One oligonucleotide is a 23 mer(5′-CCTTGCATATTAACATAACTCGC-3′) and the other oligonucleotide is a 20mer (5′-AAGTCGCCCTATTCAGACTC-3′). A comparison of PTI-1 with GenBankindicates that the 3′-part of this gene (630 to 2,123 bp) has 97%homology to human elongation factor 1-alpha. The 5′-part of this gene(1-629 bp) does not show any homology to known eucaryotic genes.

Comparison of PTI-2 with Genbank indicates that it has 86.9% identity in1356 bp overlap with Mycoplasma floccular 16S ribosomal RNA and 23Sribosomal RNA genes, but no homology to any previously identifiedeucaryotic genes.

Prostate Tumor Inducing Gene-3 (PTI-3): To identify genes specificallyexpressed in CREF-Trans 6:4 NMT (transformed by DNA from LNCaP cells).,but not CREF-Trans 6 cells applicants have used the differential RNAdisplay procedure. This approach resulted in the identification of a 214bp DNA fragment in CREF-Trans 6:4 NMT that was not present in CREF-Trans6 cells (PNAS paper). Northern blotting indicates that this 214 bp DNAis expressed in CREF-Trans 6:4 NMT and LNCaP cells, but not inCREF-Trans 6 cells. A 20 mer oligonucleotide with the sequence5′-AACTAACTGGAGGACCGAAC-3′ within this 214 bp DNA fragment was used toobtain extended sequences beyond the 214 bp DNA using the RACE method. AcDNA from LNCaP cells was synthesized with oligodT. To the 3′-end apolydC was added by terminal deoxynucleotide transferase. When theanchor primer (using the protocol of the GIBCO-BRL 5′ RACE kit) and theabove 20 mer were used to perform PCR amplification of cDNA from LNCaPcells, a 1.8 Kb DNA fragment containing a partial sequence of the 214 bpDNA was obtained. This 1.8 Kb DNA fragment displays the same Northernblotting pattern as does the unique 214 bp sequence.

The 1.8 Kb DNA fragment was cloned into PCR™ II vector by using the TAcloning kit (Invitrogen). The sequence of this 1.8 Kb DNA was determinedby Sanger's method (Sequenase kit, version 2.0 USB). The 1.8 Kb DNAcontains a partial sequence of PTI-1/3. The 5′ and 3′ end of PTI-3 generemains to be confirmed. The insert of PTI-3 1.8 Kb insert can berecovered from the PCR™ II vector by digestion with EcoRI. A comparisonof the sequence of PTI-3 with Genbank data base indicates that this genehas 87% identity in 1858 bp overlap with Mycoplasma floccular 16Sribosomal RNA and 23S ribosomal RNA genes, and it has 89.8% identity in1858 bp overlap with Mycobacterium hyopneumoniae 23S ribosomal RNA gene.

Prostate Carcinoma Tumor Antigen Gene-1 (PCTA-1): Evidence that tumorderived CREF-Trans 6 cells transfected with LNCaP DNA encode geneticinformation related to human prostate cancer has been obtained using anapproach termed surface epitope masking (SEM). The SEM procedureinvolves the selective blocking of surface antigens present in agenetically engineered cell (referred to as a “tester”) with high-titerpolyclonal antibodies against the untransfected parental cell (referredto as a “driver”). Surface-epitope-masked tester cells are injected intoBALB/c mice, immune spleen cells are then taken from these mice and theyare fused with myeloma cells. This process results in the efficientgeneration of hybridomas that secrete monoclonal antibodies (MAbs) thatreact with cell-surface antigens on transfected tester cells and withadditional cell types that express the same surface molecules. LNCaPtransfected tumor-derived CREF-Trans 6 cells, CREF-Trans 6: 4 NMT, havebeen used as a tester cell line. The SEM procedure was applied resultingin the development of hybridomas producing MAbs reacting with tumorassociated antigens (TAAs) on the surface of the original LNCaP cellline used to obtain human prostatic carcinoma DNA, primary and secondarynude mouse transfectants derived from tumors and two additional humanprostatic carcinoma cell lines (DU-145 and PC-3). These MAbs aredesignated Pro 1.1 to 1.5. Specific MAbs also display reactivity to twohuman breast carcinoma cell lines (MCF7 and T47D). However, they do notreact with normal human skin fibroblasts (NHSF-1), two colon carcinomacell lines (WiDr and LS174T), two human melanoma cell lines (H0-1 andMeWo) or a human glioblastoma multiforme cell line (GBM-18).Immunoprecipitation analyses of ³⁵S-methionine labeled cell extractswith PCTA-1 Pro 1.5 MAbs indicate that primary and secondary nude mousetumor-derived LNCaP-transfected CREF-Trans 6 cells, LNCaP and DU-145cells contain an approximately 42 kDa protein that is not present inuntransfected CREF-Trans 6 or additional human tumors (includingmelanoma and glioblastoma multiforme) These results indicate that a geneencoding human prostatic carcinoma (and possibly breast carcinoma) TAAshas been transferred and is now expressed in CREF-Trans 6 cells.

To identify the gene encoding PCTA-1l, a SEM-derived MAb (Pro 1.5) wasused to screen an LNCaP cDNA expression library (PicoBlueImmunoscreening Kit, STRATAGENE). mRNA was isolated from LNCaP cellsafter passage of total RNA through an oligo-dT column (GIBCO). LNCaPcDNA libraries were constructed in the Uni-ZAP vector (Stratagene).Screening of the cDNA library was performed as follows: (1) SURE hostcells were plated on fifteen 150 mm×15 mm NZY plates with 6.5 ml of topagar (˜2×10⁴ plaques/plate); (2) After 3.5 hr incubation at 42° C.,nitrocellulose filters soaked with 10 mM IPTG solution were applied tothe plates and plaques were lifted; (3) The filters containing theplaque lifts were washed 3 or 4× with TBST (20 mM Tris-HCl pH7.5, 150 mMNaCl, 0.05% Tween-20) and soaked in blocking solution (1% BSA in TBS [20mM Tris-HCl pH 7.5, 150 mM NaCl)] for 1 hr at room temperature; (4) Thefilters were then transferred to fresh blocking solution containing Pro1.5 ascites (1:500 dilution) followed by incubation for 3 hr at roomtemperature with gentle rocking; (5) Filters were washed 4× with TBSTbuffer; (6) The filters were transferred into fresh blocking solutioncontaining Ab-AP conjugate (1:2000 dilution) and incubated for 1 hr atroom temperature; (7) The filters were washed 4× with TBST and placed ina developing solution containing 0.3 mg/ml NBT (nitro blue tetrazolium,0.15 mg/ml of BCIP (5-bromo-4-chloro-3-indolyl phosphate), 100 mMTris-HCl pH 9.5, 100 mM NaCl, 5 mM MgCl₂); and (8) The reaction wasterminated with stop solution containing 20 mM Tris-HCl, pH 2.9 and 1 mMEDTA) and the filters were dried. In the first round of screening withPro 1.5, only 2 positive clones were obtained from over 3×10⁶ (15×2×10⁴)colonies. Screening was performed a second time and clones were isolatedand characterized. Using this approach antibody-positive clones wereidentified that contain a cDNA insert of ˜3.8 Kb. Sequence analysis ofPCTA-1 indicates no homology to previously identified genes. The 5′region of PCTA-1 is homologous to several expressed sequence tags[including Homo sapiens partial cDNA sequence clone HEC077, clonec-zvh01, clone hbc1127 (3′ end), clone hbc1208 (5′ end) and clonehbc1074 (3′ end)) (see below). In vitro translation in a rabbitreticulocyte lysate system, with and without immunoprecipitation withPro 1.5, indicate the presence of an approximately 36 kDa protein. Theseobservations indicate that the PCTA-1 cDNA encodes a protein that is theputative tumor associated antigen present on prostate cancer cellsidentified using the SEM approach. Comparison of PCTA-1 with Homosapiens cDNA clones Homo sapiens partial cDNA sequence hbc hbc 1127 1208hbc1074 Identity PCTA-1 HEC077 c-zvh01 (3′ end) (5′ end) (3′ end) 94.9%2123/1732 1-395   99% 2118/1858 1-261 88.8% 3853/3562 1-290 93.5%2630/2818 1-186   84% 2728/2825 1-98

(1) SEM-Derived MAbs Specific for the Multidrug Resistance (MDR)P-Glycoprotein encoding an 170.000 Molecular Weight Cell SurfaceTransport Protein: To develop MAbs specific for the P-glycoproteinmediating MDR, CREF-Trans 6 cells were transfected with a human MDR-1gene and cells resistant to colchicine were isolated. These MDR clonescontain the MDR gene, express MDR mRNA and are cross-resistant totoxicity induced by several chemotherapeutic agents. MDR-CREF-Trans 6cells were coated with CREF-Trans 6 polyclonal antibodies, injected intoBALB/c mice, spleens were isolated and used to from hybridomas.Hybridomas secreting MAbs specific for MDR-CREF-Trans 6 cells wereisolated. These SEM-derived MAbs react with MDR-CREF-Trans 6 cells asdemonstrated by fluorescence activated cell sorter (FACS) analysis,confirming their interaction with epitopes of the P-glycoproteinexpressed on the cell surface. In addition, human breast carcinoma(MCF-7) cells transfected with the same MDR-1 gene and displaying theMDR phenotype also react with the SEM-derived MAbs. In contrast, non-MDRparental MCF-7 cells do not react with these MAbs. These resultsindicate that the SEM approach can be used to develop MAbs specific fordefined cell surface-expressed molecules. (Full details in our JNCImanuscript—Shen, Su, Olsson, Goldstein & Fisher).

(2) SEM-Derived MAbs Specific for the Human Leukocyte Interferon α(IFN-α) Receptor: To develop MAbs specific for the Human IFN-α receptor,CREF-Trans 6 cells were transfected with a human IFN-α receptorexpression vector and clones expressing the receptor were isolated.These clones interacted with labeled IFN-α, whereas non-transfectedCREF-Trans 6 cells do not react with IFN-α. These results providefurther documentation of the effectiveness of the SEM approach inproducing MAbs specific for defined cell surface-expressed molecules.

(3) SEM-Derived MAbs Specific for the Human Immune Interferon (IFN-γ)Receptor: To develop MAbs specific for the Human IFN-γ receptor,CREF-Trans 6 cells were transfected with a human IFN-γ receptorexpression vector and clones expressing the receptor were isolated.These clones interacted with labeled IFN-γ, whereas non-transfectedCREF-Trans 6 cells do not react with IFN-γ. These results providefurther documentation of the effectiveness of the SEM approach inproducing MAbs specific for defined cell surface-expressed molecules.

(4) SEM-Derived MAbs Reacting with Human Prostate Carcinomas: Todetermine if CREF-Trans 6 cells containing a putative human prostatetumor inducing gene(s), CREF-Trans 6:4 NMT, display tumor associatedantigens (TAAs) also expressed on human prostate carcinoma cells, theSEM approach has been used. This procedure and the experimental resultsusing the CREF-Trans 6:4 NMT clone is described in our JNCI manuscript(Shen et al., JNCI 86: 91-98, 1994). The SEM-derived Pro MAbs (Pro 1.1,Pro 1.2, Pro 1.3, Pro 1.4 and Pro 1.5) display reactivity with LNCaPcells as well as two additional human prostate carcinomas, DU-145 andPC-3. Specific Pro MAbs also display surface reactivity with two humanbreast carcinoma cell lines, T47D and MCF-7. These MAbs are now beingtested for reactivity using in situ immunohistochemistry with sectionsobtained from patients with prostate cancer. The ability to generatethese Pro MAbs by SEM indicate that this approach can also be used toproduce MAbs specific for cell surface expressed molecules of unknownorigin. The Pro 1.4 MAbs have also been used in combination withexpression cloning and human prostate carcinoma library screening toidentify and clone the gene encoding the specific TAAs, PCTA-1.

(5) SEM-Derived MAbs Reacting with Human Breast Carcinomas: To determineif CREF-Trans 6 cells containing a putative human breast carcinoma tumorinducing gene(s), CREF-Trans 6:T47D NMT, display TAAs also expressed inhuman breast carcinoma cells, the SEM approach has been used. Thisapproach resulted in the development of SEM-derived Br-car (breastcarcinoma) MAbs (4.2.1 and 5.2.4) that react with T47D and MCF-7 humanbreast carcinoma cell lines. In situ immunohistochemistry (total of 10samples) indicate that the SEM-derived Br-car MAbs also react withcarcinoma sections from patients with ductal and medullary breastcarcinomas (FIG. 12). These MAbs are negative in sections of humanmelanoma and a small cell lung carcinoma (FIG. 12). The ability togenerate these Br-car MAbs by SEM provide additional evidence that thisapproach can be used to produce MAbs specific for cell surface expressedmolecules of unknown origin.

Potential Applications for SEM-Approach and SEM-Derived MAbs: (A) TheSEM-approach represents a general strategy for producing MAbs specificfor molecules expressed on the cell surface. These can include, but arenot limited to, novel TAAs, growth factor receptors, T-cell reactiveepitopes, cell surface antigens (representing different developmentalstages), surface expressed oncogene products, viral encoded proteinsfound on the cell surface, surface antigens expressed as a function oftumor progression (e.g., antigens associated with benign disease andmetastatic disease), surface antigens eliciting reactivity withnon-specific immunoreactive cells (i.e., NK cells and macrophages) andsurface antigens eliciting autoimmune diseases (diagnostic andtherapeutic applications); (B) SEM-derived MAbs can be used for in situimmunohistochemistry to identify specific cell surface expressedmolecules, including growth factor receptors, cell surface antigens,surface expressed oncogene products, TAAs and viral encoded proteinsfound on the cell surface (diagnostic applications); (C) SEM-derivedMAbs can be used to target toxins and radionuclides to tumor cells(therapeutic applications); (D) SEM-derived MAbs with high reactivitytoward specific clinically relevant target molecules can be used todevelop chimerized (human-mouse) and humanized MAbs for both diagnosticapplications and therapeutic applications in humans; (E) SEM-derivedMAbs can be used to clone genes encoding TAAs and additional cellsurface expressed molecules of unknown structure. These genes can thenbe used for diagnostic applications and ultimately therapeuticapplications; (E) SEM-derived MAbs can be used to identify potentiallyimportant TAAs. Once appropriate genes and antigens are identified theycan be used as part of a strategy to vaccinate against specific TAAs(therapeutic applications).

Fourth Series of Experiments

The selective production of monoclonal antibodies (MAbs) reacting withdefined cell surface expressed molecules is now readily accomplishedwith an immunological subtraction approach, surface-epitope masking(SEM). Using SEM, prostate carcinoma (Pro 1.5) MAbs have been developedthat react with tumor associated antigens expressed on human prostatecancer cell lines and patient-derived carcinomas. Screening a humanLNCaP prostate cancer cDNA expression library with the Pro 1.5, MAbidentifies a gene, prostate carcinoma tumor antigen-1 (PCTA-1). PCTA-1encodes a secreted protein of ˜35 kDa that shares ˜40% sequence homologywith the N-amino terminal region of members of the S-(soluble)galactose-binding lectin (galectin) gene family. Specific galectins arefound on the surface of human and murine neoplastic cells and have beenimplicated in tumorigenesis and metastasis. Primer pairs within the 3′untranslated region of PCTA-1 and reverse transcription-PCR demonstrateselective expression of PCTA-1 by prostate carcinomas versus normalprostate and benign prostatic hypertrophy. These findings document theuse of the SEM procedure for generating MAbs reacting with tumorassociated antigens expressed on human prostate cancers. The SEM-derivedMAbs have been used to expression clone the gene encoding this humantumor antigen. The approaches described, SEM combined with expressioncloning, should prove of wide utility for developing immunologicalreagents specific for and identifying genes relevant to human cancer.

Production of MAbs reacting with antigens present on the surface oftumor cells, but displaying restricted expression on normal cells, isoften a difficult and inefficient process (1-3). A procedure based onimmunological subtraction, SEM, has been developed that in principle canobviate many of the limitations preventing efficient MAb developmenttoward molecules expressed on the cell surface (3, 4). SEM is based onthe selective blocking of antigens on a genetically modified targetcell, i.e., tester, with polyclonal antibodies produced against the sameunmodified cell line, i.e., driver. Antigen blocked cells are injectedinto BALB/c mice and sensitized spleen cells are removed, fused to NS1myeloma cells and hybridomas secreting reactive MAbs are isolated (3).The SEM approach has been successfully used for a number of applicationsresulting in the production of MAbs specific for surface expressedmolecules with known and unknown functions (3, 4). These include MAbsreacting with the 170,000 M_(r) human multidrug resistance protein(P-glycoprotein) and the human interferon gamma receptor (3, 4). Inaddition, SEM has been used to produce MAbs, Pro 1.1 to 1.5, reactingwith TAAs expressed on appropriate genetically modified CREF-Trans 6 andhuman prostate carcinoma cell lines (3).

An improved procedure has been developed for identifying and cloningdominant acting oncogenes, termed rapid expression cloning (5, 6). Thisapproach involves transfecting high molecular weight human tumor DNAinto a new acceptor cell line, CREF-Trans 6, selecting cells expressinggenes inducing tumors in nude mice and using molecular biologicalapproaches, such as differential RNA display, to clone the putativeoncogene (5, 6). Tumor-derived CREF-Trans 6 cells have also provenuseful for identifying TAAs expressed on the cell surface of cancercells serving as the initial source for transforming DNA (5, 6).Expression cloning of cDNAs using antibodies reacting with their encodedproteins represents a direct means of identifying and cloning functionalgenes (2). This approach has now been used to identify and clone a gene,prostate carcinoma tumor antigen-1 (PCTA-1), encoding TAAs recognized bythe MAb Pro 1.5. Sequence analysis indicates that PCTA-1 consists of acDNA of 3.8 Kb with no homology to previously identified genes. Someoverlapping homologies are detected with several small noncontiguouspartial cDNA sequences (of less than 500 nucleotides) previouslyidentified as expressed human sequence tags (7). Analysis of proteinstructure indicates that PCTA-1 has ˜40% homology to specific structuraldomains of the S-lectin family of galactose-binding lectin proteins, thegalectins (8-10). These highly homologous proteins include acarbohydrate-binding 35 kDa protein CBP35 expressed on NIH-3T3fibroblasts, a 34 kDa galactose-binding surface antigen present onmetastatic murine tumors, a 31 kDa galactose-binding surface protein onmetastatic human tumors, a 30 kDa carbohydrate-binding protein CBP30found on baby hamster kidney cells, rat and human lung 29 kDagalactose-binding lectins, an IgE-binding protein of rat basophiliccells and a 32 kDa surface antigen Mac-2 found onthioglycollate-elicited murine macrophages (6-8). The roles of theS-lectin (galectin) proteins are diverse and impinge upon importantbiological processes including cell signaling, proliferative control,cell adhesion and cell migration (8-10). In this context, thedemonstration that PCTA-1 encodes a protein with homology to theS-lectins (galectins) places this molecule in a pivotal positionrelative to human prostate cancer development and evolution.

Materials and Methods

Cell Lines

The LNCaP cell line was derived from metastatic deposits from a patientwith advanced prostate cancer (11). CREF-Trans 6 and LNCaPDNA-transfected nude mouse tumor-derived CREF-Trans 6 cells, CREF-Trans6:4 NMT, were isolated as described previously (5). The hormoneindependent prostatic carcinoma cell lines DU-145 and PC-3 were obtainedfrom the American Type Culture Collection. Conditions for growing thevarious cell types were as described previously (5, 6, 11).

cDNA Library Construction, Expression Cloning and Sequencing

An LNCaP cDNA library was constructed in the Uni-ZAP XR vector(Stratagene®) (6, 12, 13). The cDNA library was screened using the Pro1.5 MAb following the protocol in the picoBlue Immunoscreening Kit(Stratagene). Host bacterial cells (SURE) were plated on fifteen150-mm×15-mm NZY plates to yield ˜20,000 plaques/plate. After 3.5 hrincubation at 42° C., nitrocellulose filters soaked in 10 mM IPTGsolution were added to the top of colonies for plaque lifts. The filterswere washed 3 to 4× with TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05%Tween-20) and soaked in blocking solution [1% BSA in TBS (20 mM Tris-HClpH 7.5, 150 mM NaCl)] and incubated for 1 hr at room temperature. Thefilters were then transferred into fresh blocking solution containingPro 1.5 ascites (1:500 dilution) and incubated for 3 hr at roomtemperature with gentle agitation. After washing 4× with TBST buffer,the filters were transferred into fresh blocking solution containingAb-AP conjugate (1:2000 dilution) and incubated for 1 hr at roomtemperature. Positive colonies were identified by developing the filtersin a solution containing 0.3 mg/ml NBT (nitroblue tetrazolium), 0.15mg/ml of BCIP (5-bromo-4-chloro-3-indolyl phosphate), 100 mM Tris-HCl pH9.5, 100 mM NaCl and 5 mM MgCl₂. The reaction was terminated by addingstop solution (20 mM Tris-HCl pH 2.9 and 1 mM EDTA. Positive 1 clonescontaining PCTA-1 were isolated. The complete sequence of PCTA-1 wasobtained using the Sanger method (14). A series of oligonucleotidessynthesized from both sides of the PCTA-1 insert in the pBluescriptvector were used as primers. Sequence analysis was verified using anApplied Biosystems (Model 373A, Version 1.2.1) sequencer.

In Vitro Translation of PCTA-1

The plasmid DNA containing PCTA-1 was linearized by digestion with XmaIII and used as a template to synthesize mRNA using the mCAP mRNAcapping kit (Stratagene). In vitro translation of PTI-1 was performedusing a rabbit reticulocyte lysate translation kit with conditions asdescribed by GibcoBRL (MD) (6).

Preparation of Mouse Polyclonal Antibodies and SEM

CREF-Trans 6 polyclonal antibodies were prepared as described (3). TheCREF-Trans 6 polyclonal antibodies were used to coat CREF-Trans 6:4 NMTcells by the SEM approach resulting in the production of hybridomassecreting Pro 1.5 MAbs (3).

Fluorescence Cell Staining and Immunostaining of Tissue Sections withPro 1.5 and PSA

Fluorescence staining using Pro 1.5 MAb used previously describedprotocols (15). Tissue sections were prepared from fresh tissues frozenin liquid nitrogen. Serial sections for several tissues were used toprepare RNA for RT-PCR (6). Staining of tissue sections used standardprotocols (Super Sensitive Detection System (BioGenex). Briefly,sections were fixed in acetone, blocked with 3% H₂O₂ for 7 min at roomtemperature andincubated for 20 min at room temperature in PBScontaining 3% lamb serum. Sections were then incubated with Pro.1.5(1:100) or PSA (1:200) (DAKO Corporation) for 45 min at roomtemperature. Samples were incubated with biotinylated secondary antibodyand alkaline phosphatase conjugated steptavidin in PBS. Prior to eachincubation step, sections were washed 3 to 5× with PBS. Reactivity wasdetected by adding DAB and counterstaining with Hematoxylin.

Immunoprecipitation Analysis

Cells were labeled with ³⁵S-methionine and cell lysates were analyzedfor PCTA-1 protein levels by immunoprecipitation analysis with Pro 1.5MAbs as previously described (3, 16). Secreted PCTA-1 was detected bylabeling cells for 4 hr with ³⁵S-methionine, growing cells for anadditional 24 hr in the absence of label, collecting the medium,concentrating the medium and performing immunoprecipitation analysiswith Pro 1.5 MAbs.

RNA Preparation and RT-PCR

Total cytoplasmic RNA was isolated from logarithmically growing cellcultures as previously described (6, 13). Tissue samples from normalprostates and patients with prostatic carcinomas or BPH were frozen inliquid nitrogen and RNA was isolated using the TRIzol reagent asdescribed by GibcoBRL (MD). Tissue samples were supplied by theCooperative Human Tumor Network (CHTN). Three samples of normal prostatewere obtained from autopsies of males <40 years of age. All tissues werehistologically confirmed as normal, BPH or carcinoma of the prostate(6). RT-PCR using primer pairs for PCTA-1, PSA and GAPDH was performedas described previously (6, 16).

Experimental Results

Production of Pro 1.5 MAbs by SEM and reactivity of Pro 1.5 and PSA withnormal prostate, BPH and prostate carcinomas

The SEM approach of blocking antigenic epitopes on nude mousetumor-derived LNCaP DNA transfected CREF-Trans 6 cells with CREF-Trans 6polyclonal antibody prior to injection into mice was used to producehybridomas secreting the Pro (prostate carcinoma) series of MAbs (3).The Pro MAbs can detect by in situ fluorescence microscopy andfluorescence activated cell sorter (FACS) analysis the surfaceexpression of tumor associated antigens on LNCaP-transfected primary(CREF-Trans 6:4 NMT) and secondary tumor-derived CREF-Trans 6 cells andLNCaP, DU-14.5 and PC-3 human prostate cancer cell lines (3) (FIGS. 16A,16B, 16C and data not shown). In contrast, Pro 1.5 does not react usingfluorescence microscopy or FACS with CREF-Trans 6 cells (3). Thestaining pattern with Pro 1.5 in human prostate carcinoma cells isirregular with microclusters, as previously observed with MAbs reactingwith specific galectins (10, 17, 18) (FIGS. 16A, 16B, 16C).Immunoprecipitation analysis identifies an ˜35 to 42 kDa protein inlysates from LNCaP-transfected primary and secondary tumor-derivedCREF-Trans 6, LNCaP, DU-145 and PC-3 cells (3) (FIG. 1D and data notshown). Of the prostate cancer cell lines studied, the PC-3 clonecontains the lowest quantities of cell associated, surface expressed(FACS) and secreted PCTA-1 protein.

To determine if Pro 1.5 MAbs can react with patient-derived prostatecancer specimens, frozen sections were prepared from normal (males <40years of age), BPH and carcinomas of the prostate. Normal prostatedisplays limited reactivity with Pro 1.5, whereas PSA readily stainsnormal prostate epithelial cells (FIG. 17). As expected, PSA also stainsprostate cells in tissue sections of BPH and prostate carcinomas. MAbPro 1.5 reacts strongly with prostate carcinoma cells present in frozentissue sections, but not with adjacent benign glands or tissue sectionscontaining normal prostate or BPH epithelium. However, some reactivitywith Pro 1.5 is found in prostatic intraepithelial neoplasia (PIN)-These studies indicate that the Pro 1.5 MAb can distinguish betweenprostate carcinoma and PIN versus normal prostate epithelial cells andBPH. In this context, Pro 1.5 provides a discriminatory capacity fordetection of cancer of the prostate that exceeds that of the nonspecificprostate epithelial cell marker PSA.

Expression Cloning of PCTA-1 Using SEM-Derived Pro 1.5 MAbs.

To identify the gene encoding the TAAs identified on human prostatecancer cells by MAb Pro 1.5 an antibody expression cloning strategy wasused. An LNCaP cDNA library was constructed in the Uni-ZAP XR vector(Stratagene®) and screened with the Pro 1.5 MAb (12, 13). This approachresulted in the identification of a 3.8 Kb cDNA clone referred to asPCTA-1. In vitro protein translation of the PCTA-1 cDNA results in a 317aa protein of ˜35 kDa (data not shown). Although the DNA sequence ofPCTA-1 displays no homology to previously identified genes, proteincomparison indicates that the PCTA-1 protein is ˜40% homologous tospecific regions of the S-lectin family of galactose-binding lectinproteins, the galectins (FIGS. 18A, 18B). Several types of proteinsequence homologies are found between PCTA-1 and the other galectins.Stretches of identical amino acids are found in PCTA-1 and both thesmall ˜14 kDa and larger ˜29 to 34 kDa galectins, e.g., HFNPRF, IVCN andWG. These amino acids are well conserved and are found in galectinsisolated from diverse species, including eel, chicken, mouse, rat,bovine and human (FIG. 18B) (10, 19, 20). They are important structuralcomponents of the galectins and may mediate galectin binding to itsputative ligands (10). Of potential interest is the replacement inPCTA-1 of two amino acids normally present in the conserved regions ofmost of the galectins, i.e., the substitution of an I for an R and a Tfor an F (FIG. 18B). A number of similar positioned amino acids arefound in PCTA-1 and the larger ˜29 to 34 kDa human and mouse galectins,that are not present in the ˜14 kDa galectins isolated from most otherspecies, e.g., DVAF and WGREE (FIG. 18B). In addition, a number of aminoacids are distinct for PCTA-1 versus the other similar positioned aminoacids that are found in the human galectin-3 L29 and Human L-31proteins, including RA, KRAG, LI, and ITYDT. The similarities in regionsof conserved structure between PCTA-1 and the galectins suggest thatthey will share a number of overlapping properties. However, thenumerous amino acid changes observed in PCTA-1, in both the conservedregions and throughout the remainder of this protein, can be expected toimpact on the structure and affect the properties of the PCTA-1 protein.

Expression of PCTA-1 in Cell Lines and Normal Prostate, BPH and ProstateCarcinomas

After obtaining the sequence of PCTA-1, studies were performed todetermine if primers for specific regions of this gene could beidentified that would permit detection of RNA expression by RT-PCR.Using primers located between bp 3010 and bp 3423 in the 3′ untranslatedregion, PCTA-1 expression is apparent in LNCaP DNA-transfectedtumor-derived CREF-Trans 6, LNCaP and DU-145 cells, but not inuntransfected CREF-Trans 6 cells (FIG. 19). PCTA-1 expression alsooccurs in seven of seven patient-derived prostate carcinomas, one offour BPH and one of four putative normal prostate tissue samples (FIG.19). In the one BPH sample displaying PCTA-1 expression, histologicalanalysis indicated the presence of epithelial atypia consistent withPIN. In the one putative normal prostate tissue sample expressingPCTA-1, no tissue was available for histological analysis. However,since this tissue was obtained from a 60 year-old male, it is possiblethat this tissue may have contained clinically unsuspected prostatedisease. The same RNA samples were tested for prostate specific antigen(PSA) expression (FIG. 19). PSA RNA was present in LNCaP cells and allof the prostate tissue samples, but not in CREF-Trans 6, LNCaPDNA-transfected tumor-derived CREF-Trans 6 or DU-145 cells. Aspredicted, all samples were positive for expression of the housekeepinggene GAPDH (FIG. 19). Although the sampling size is small, these resultsindicate that PCTA-1 expression may be restricted to prostate carcinomasand subsets of BPH displaying early stages of cancer, i.e., PIN. Incontrast, PCTA-1 expression is not evident in normal prostate or BPH.

Secretion of PCTA-1 by human prostate carcinoma cell lines. Manygalectins are externalized by non-classical secretory mechanisms that donot involve a typical secretion signal peptide (10, 17, 18). In order todetermine if PCTA-1 is secreted by Pro 1.5 MAb positive cells, targetcells were labeled for 4 hr with ³⁵S-methionine, the label was removedand cells were washed three times in methionine-free medium and thenincubated for an additional 18 to 24 hr in complete medium withoutlabel. The conditioned medium (CM) was collected, contaminating cellswere removed, the CM was concentrated and immunoprecipitated using thePro 1.5 MAb (FIG. 16C). A protein of ˜35. to 42 kDa was present in CMfrom CREF-Trans 6:4 NMT, LNCaP, DU-145 and PC-3, but not from CMobtained from CREF-Trans 6 cells (FIG. 16C). Immunoprecipitationanalysis of ³⁵S-methionine labeled cell lysates from the same cell typesindicate a similar pattern of PCTA-1 expression, i.e., present inCREF-Trans 6:4 NMT and human prostate carcinoma cell lines, but not inuntransfected CREF-Trans 6 cells (FIG. 16C). As previously found usingFACS analysis with the Pro MAbs, PC-3 cells produced the lowest levelsof cell-associated and secreted PCTA-1. These results demonstrate thatPCTA-1 is shed from prostate cancer cells. In this context, monitoringPCTA-1 protein in the circulation might prove beneficial as a diagnosticmarker for prostate cancer.

Experimental Discussion

The early detection of prostate cancer and the accurate prediction ofits clinical course is not possible using current methodologies.Contemporary approaches, including physical examination, tissue biopsy,monitoring serum PSA levels, and ultrasound and bone scans do not insureearly prostate cancer detection and are of only limited value inpredicting disease progression. Of immense value for the accuratediagnosis and potentially for the therapy of human prostate cancer isthe identification of immunological and genetic reagents displaying theappropriate specificity that will permit a clear distinction betweenprostate carcinoma versus normal prostate and BPH. By using a number ofinnovative strategies, including rapid expression cloning, SEM andantibody expression cloning, we have produced MAbs reacting with TAAsdifferentially expressed on human prostate carcinoma cells versus normalprostate and BPH and have cloned the gene. PCTA-1 that encodes thisprotein. PCTA-1 is expressed in invasive prostate carcinoma and earlyprostate cancer, PIN, but not in histologically confirmed normalprostate or BPH. The PCTA-1 encoded TAAs are detected on the surface ofprostate cancers and are shed by prostate carcinoma cells. Theseattributes should allow the direct use of the Pro series of MAbs and thePCTA-1 gene for diagnostic applications. If appropriate specificity ofthe PCTA-1 gene is found using a larger tissue sampling, this gene mayalso prove useful for designing gene-based strategies for the therapy ofprostate cancer.

The PCTA-1 protein retains a number of conserved structural motifs thatare found in most members of the galectin gene family (10, 19, 20).These conserved regions are present in species as diverse as eel, mouse,rat and human (10). On the basis of the DNA sequence of PCTA-1 and itsencoded protein, PCTA-1 is a new member of the galectin gene family,galectin-8, that may contribute to the cancer phenotype of humanprostate carcinomas. The galectins display wide tissue distribution,clear developmental regulation and differential levels in specifictissues, supporting the hypothesis that they contribute to manyphysiologically important processes in mammalian cells (10). Of directrelevance to cancer, is the finding that the galectins, as well as theselectin subgroup of C-type lectins (21, 22), can mediate both cell-celland cell-matrix interactions (10, 17, 23). These associations arecritical elements in mediating the metastatic spread of tumor cells(24). Moreover, experimental evidence has accumulated indicating thatgalectin-3 may play an important role in the metastatic process (17,25-29). Galectin-3 is overexpressed in human colon and gastriccarcinomas versus normal and benign tissue and elevated expression ofrecombinant L-34 in a weakly metastatic UV-2237-cl-15 mouse fibrosarcomacell line increases lung metastases in syngeneic and nude mice (17,25-29). Moreover, anti-galectin MAbs inhibit homotypic aggregation,anchorage-independent growth and experimental metastases in UV-2237subclones (30-32). Studies are in progress to determine if the PCTA-1gene and the Pro MAbs display similar properties as the clonedgalectin-3 gene and the anti-galectin MAbs, respectively. It will alsobe important to ascertain if inhibition in PCTA-1 expression, usingantisense oligonucleotides, antisense expression vectors or ribozymeapproaches, alters the tumorigenic or metastatic properties of humanprostate cancers.

Prior to SEM, no simple and direct procedure was available forefficiently generating MAbs reacting with differentially expressedsurface molecules. Conceptually, SEM involves immunological subtractionthat induces the immune system of mice to target antibody productiontoward surface molecules expressed on a genetically modified tester cellline, but not expressed or expressed in lower abundance on its' cognateunmodified driver cell line (3, 4). By using polyclonal antibodiesproduced against the driver cell line to coat (mask) epitopes on thetester cell line, enriched production of MAbs targeted toward epitopesexpressed on the surface of the tester cell type is achieved (3, 4). Inaddition to identifying TAAs present on the surface of human prostatecancer cells, Pro 1.1 to 1.5, the SEM approach has also been used withrapid expression cloning to develop MAbs reacting with human breastcarcinoma cells (data not shown). These results indicate that theseapproaches may represent an efficient strategy for identifying antigenicepitopes on human cancers. Additional applications of SEM may alsoresult in the targeted production of MAbs and the identification ofgenes associated with important physiological processes, includingcellular growth and differentiation, immunological recognition,tumorigenesis, metastasis, cellular senescence, atypical multiple drugresistance and autoimmune disease.

In summary, applicants presently demonstrate direct applications of therapid expression cloning and SEM technologies for the development ofMAbs and the cloning of the PCTA-1 gene associated with human prostatecancer. The PCTA-1 gene and the recently identified prostate tumorinducing gene PTI-1 (6) are genetic elements that can distinguishprostate cancer from normal prostate and BPH. Although further studiesare required, it appears that PCTA-1 may represent an earlier geneticchange in human prostate cancer development than PTI-1. In this context,both the Pro MAbs and the PCTA-1 gene should find direct applicationsfor prostate cancer diagnosis and staging and they may also representimportant therapeutic reagents for intervention in this pervasive andoften fatal neoplastic disease.

REFERENCES OF THE FOURTH SERIES OF EXPERIMENTS

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Fifth Series of Experiments

Rapid expression cloning and differential RNA display identified a novelprostatic carcinoma oncogene, prostate tumor inducing gene-1 (PTI-1).PTI-1 consists of a 630 bp 5′-UTR with sequence homology to Mycoplasmahyopneumoniae (5′-UTR) and a 3′ sequence encoding a truncated andmutated form of human elongation factor-1a. Screening a cDNA libraryconstructed from LNCaP human prostate cancer cells with the 5′-UTRindentifies additional PTI cDNAs with similar 5′-UTR regions, butdiffering 3′ regions. Southern hybridization analysis of genomic DNAsdemonstrates the presence of PTI 5′-UTR sequences in organisms asdiverse as bacteria, yeast and human. Using a 5′-UTR probe of PTI-1, RNAexpression occurs in cloned rat embryo fibroblast (CREF) cellstransformed by diverse acting viral oncogenes, including adenovirus type5 (Ads), a cold-sensitive host-range Ad5 mutant, T24 Ha-ras, v-src andhuman papilloma virus type-51 (HPV-51). A CREF clone containing awild-type Ad5 E1A transforming gene under the transcriptional control ofa dexamethasone (DEX)-inducible mouse mammary tumor virus promoterdemonstrates a direct relationship between induction of Ad5 E1A, the5′-UTR of PTI-1 and cellular transformation. Expression of the 5′-UTR ofPTI-1 is regulated at a transcriptional level in the DEX-inducible Ad5E1A-transformed CREF clone, as well as in v-src, Ha-ras andHPV-51-transformed CREF cells. These results indicate that morphologicaland oncogenic transformation of CREF cells correlates withtranscriptional induction of the 5′-UTR of PTI genes. Since this 5′-UTRsequence is a component of multiple cellular RNAs, genes linked to this5′-UTR may be targets for oncogenic transformation.

It is now possible to detect dominant-acting tumor-inducing oncogenes ingenomic DNAs from human cancer cell lines and primary patient-derivedtumors using the rapid expression cloning procedure with a new DNAacceptor cell line CREF-Trans 6. This approach has been used with highmolecular weight DNA from the human LNCaP prostate carcinoma cell lineto demonstrate the presence of a putative nude mouse tumor inducinggene, not detected using similar approaches with NIH-3T3 cells. Tumorsdeveloping in nude mice injected with LNCaP DNA transfected CREF-Trans 6cells were established in culture, mRNAs were compared by differentialRNA display (DD) and a novel sequence of 214 bp (representing part ofthe 5′-UTR of PTI-1) was identified and cloned. When Northern blotscontaining RNAs from various cell types were probed with the 214 bpsequence, RNA expression was detected in LNCaP transfected nude mousetumor-derived CREF-Trans 6, LNCaP, DU-145 (hormone refractile humanprosate carcinoma), NCI-H69 (human small cell lung carcinoma), T47D(human breast carcinoma), SW-480 and LS174T (human colorectalcarcinomas) cells. Screening an LNCaP cDNA library with the 214-bp DNAfragment and RACE 5′ extension resulted in the cloning of a full-length2.0-kb PTI-1 cDNA. Sequence analysis indicates that PTI-1 consists of a630-bp 5′ sequence with homology to Mycoplasma hyopneumoniae and a 3′sequence homologous to a truncated and mutated form of human elongationfactor 1-a (EF-1a). Using a pair of primers recognizing a 280-bp regionwithin the 630-bp 5′-UTR PTI-1 sequence, reverse transcription-PCRdetects PTI-1 expression in patient-derived prostate carcinomas but notin normal prostate or benign hypertrophic prostate tissue. Thesefindings indicate that PTI-1 may be a member of a class of oncogenesthat could affect protein translation and contribute to carcinomadevelopment in human prostate and other tissues.

The present study was designed to determine the potential significanceof the mycoplasmal-like 5′-UTR region of PTI-1. We demonstrate that thissequence is present in genomic DNA of both procaryotic and eukaryoticcells. In humans, tissue expression of the 5′-UTR occurs in normalskeletal muscle and colon tissue, as well as in specific carcinomas ofthe prostate, colon, lung and breast. cDNAs isolated from an LNCaP cDNAlibrary indicate that the 5′-UTR is associated with multiple distinct 3′regions, suggesting that this sequence is part of a PTI multigenefamily. Expression of the 5′-UTR of PTI-1 correlates with transformationin rat embryo fibroblast cells by diverse DNA viruses. The induction ofthe 5′-UTR in oncogene-transformed rat embryo cells occurs by atranscriptional mechanism. These observations suggest that cDNAscontaining the 5′-UTR of PTI-1 may be targets for transcriptionalactivation occurring during oncogenic transformation.

1-127. (canceled)
 128. A purified mammalian Prostate Tumor InducingGene-1 protein.
 129. The purified mammalian Prostate Tumor InducingGene-1 protein of claim 128, wherein the protein comprises the aminoacid sequence of SEQ ID NO:17.
 130. An antibody capable of specificallybinding to the mammalian Prostate Tumor Inducing Gene-1 protein of claim128.
 131. The antibody of claim 130, wherein the antibody is amonoclonal antibody.
 132. A composition comprising an antibody of claim130 and a pharmaceutically acceptable carrier.
 133. The antibody ofclaim 130 coupled to a cytotoxic agent.
 134. The antibody of claim 133,wherein the cytotoxic agent is a radioisotope or a toxin.
 135. Animmunoassay for detecting the presence of a mammalian Prostate TumorInducing Gene-1 protein in a biological sample comprising the steps of:a) contacting the biological sample with the antibody of claim 130 underconditions permitting the formation of a complex between said antibodyand the mammalian Prostate Tumor Inducing Gene-1 protein; and b)detecting said complex, wherein detection of said complex indicates thepresence of mammalian Prostate Tumor Inducing Gene-1 protein in abiological sample.
 136. A mammalian Prostate Tumor Inducing Gene-1protein bound to the antibody of claim
 130. 137. A method for detectingcancer cells in a sample comprising detection of the expression ofProstate Tumor Inducing Gene-1 in the sample, comprising the steps of:a) contacting the sample with an antibody capable of specificallyrecognizing PTI-1 protein under conditions permitting the formation of acomplex between the PTI-1 protein and the antibody; and b) measuring thecomplex formed, thereby detecting cancer cells in the sample.
 138. Themethod of claim 137, wherein the antibody is a monoclonal antibody.